# Overview Parhelia Omni-Stainer™ system is a precise, cost-effective tissue stainer designed to deliver consistent results while offering substantial savings in reagents and time. Parhelia Omni-Stainer™ system utilizes capillary action to provide smooth laminar flow of tissue staining reagents for slide mounted ([Omni-Stainer S12](https://parheliabio.com/product/omni-stainer-s12-automation-bundle/?utm_source=gitbook\&utm_medium=gitbook)) and coverslip mounted ([Omni-Stainer C12](https://parheliabio.com/product/omni-stainer-s12-automation-bundle/?utm_source=gitbook\&utm_medium=gitbook)) samples, yielding reproducible results with huge reagent and time savings. {% embed url="" %} [Parhelia Omni-Stainer™ ](https://parheliabio.com/)Promo Video {% endembed %} ## Capillary gap liquid exchange

Parhelia Omni-Stainer™ Capillary Action
100μl of blue dye dispensed into S12 flow cell via Opentrons OT-2

With capillary gap staining, the sample rests within a staining chamber, incubating in a thin capillary layer of liquid, thus resulting in a uniform exposure of tissue to the reagents.

Cross-Sectional View of Slide Mounted Sample with Parhelia Omni-Stainer™ Cover Pad

Cross-Sectional View of Coverslip Mounted Sample with Parhelia Omni-Stainer™ Support Pad

The liquid layer is held in place by the capillary force and won't spontaneously flow out of the chamber even if the sample is held vertically. However, the "magic" happens, when a new liquid is applied to the top edge of the staining chamber: the new liquid instantly displaces the one that is currently occupying the chamber.

Capillary Action Figure

{% hint style="info" %} Questions?Try asking Lens (top right), a semantic search AI that scans this documentation and gives you a simple, semantic answer with clickable references if you want to dive deeper. ![](/files/5YNbArCuIZEs5PMgABaa) {% endhint %} {% hint style="info" %} If you would prefer to ask a human your question, or share any comments, or concerns with your Parhelia products please email for information, for sales, or for product support for existing customers. {% endhint %} # Quick Start: Omni-Stainer™ Bundles Manual and Automated Set Up ## Recommended for beginners:
Documentation PagePurpose
/pages/2dJO3qEqJrDxJdolHzmoGet to know what this is all about
/pages/MxZdTK5EFraKjPXYeAwdGet acquainted with the Box Contents of your specific product bundle
/pages/yLBsmygr2ER4NsQkRPDAThese consumables are required in addition to the Omni-Stainer bundle for the first runs
/pages/NmuN25IFo3T1ISCeUP2RGo here if using S12 (slide-mounted samples)
/pages/1H3JOB80iwMXtLYKepXkGo here if using C12 (coverslip-mounted samples)
/pages/xzxNw62cWQpoXqHIkjIjHow to get the automation up and running
/pages/2AiFaxsxAuEUP9KcOfyQIf you're just doing manual operations

There are S-type (S12) and C-type (C12) Omni-stainers, both have automated and manual operation options.

{% hint style="info" %} If you have any questions, comments, or concerns with your Parhelia products please email [info@parheliabio.com](mailto:undefined) {% endhint %} # Omni-Stainer™ Box Contents First, let's get acquainted with the Box Contents. {% hint style="warning" %} The blue dye bottle in our [product bundles](https://parheliabio.com/product-category/new-product-bundle/) is in a seperate bag outside of the white box. {% endhint %} ## Parhelia Omni-Stainer™ S12 Module (slide-mounted samples)
## Parhelia Omni-Stainer™ C12 Module (coverslip-mounted samples)
{% embed url="" %} Unboxing the [Omni-Stainer™ S12 – Automation Bundle](https://parheliabio.com/product/omni-stainer-s12-automation-bundle/) {% endembed %}

Parhelia Omni-Stainer™ S12 on deck in the Opentrons OT-2

# Additional materials These consumables are required in addition to a Parhelia Omni-Stainer™ Automation Bundle for the first runs {% hint style="info" %} The materials below are only required for automated operation. **For manual operation, you only need a micropipette + tips, and your samples.** {% endhint %}
Additional Parhelia labware shown above with the [Parhelia Omni-Stainer™ S12 Module Automation Bundle](https://parheliabio.com/product/omni-stainer-s12-automation-bundle/?utm_source=gitbook\&utm_medium=gitbook), in the [OT-2 robot](https://parheliabio.com/product-category/opentrons-robot-purchase/?utm_source=gitbook\&utm_medium=gitbook) with [Gen2 P300 Single-Channel](https://parheliabio.com/product/opentrons-single-channel-electronic-pipette-p300/?utm_source=gitbook\&utm_medium=gitbook) and [Opentrons 300 µl Pipette Tips](https://parheliabio.com/product/opentrons-300-ul-pipette-tip-rack-and-refill/?utm_source=gitbook\&utm_medium=gitbook) performing 10X Visium H\&E staining @ The Broad Institute. ## [Parhelia Biosciences Reagent Reservoir Starter Kit](https://parheliabio.com/product/parhelia-biosciences-reagent-reservoir-starter-kit/?utm_source=gitbook\&utm_medium=gitbook) The Starter Kit contains all the additional consumables needed to stain your samples, besides reagents, slides/coverslips, and [pipette tips](https://parheliabio.com/product/opentrons-300-ul-pipette-tip-rack-and-refill/?utm_source=gitbook\&utm_medium=gitbook), in a convenient quantity.

Parhelia Biosciences Reagent Reservoir Starter Kit contains 3 X Deep-Well 12-Trough Reservoirs, 10 X Skirted 96-Well PCR Plates, and 50 X Pierceable Sealing Films.

{% hint style="danger" %} It's crucial to avoid using unvalidated labware as it can potentially result in notable automation errors. {% endhint %} {% hint style="success" %} Obtain an [Parhelia Biosciences Reagent Reservoir Starter Kit](https://parheliabio.com/product/parhelia-biosciences-reagent-reservoir-starter-kit/?utm_source=gitbook\&utm_medium=gitbook) to get up and staining quickly! {% endhint %}
Pierceable aluminum foil seals Peelable plate seals are highly beneficial for preparing and storing reservoirs. Specifically, aluminum foil can be pierced by pipette tips, eliminating the need to peel off the seal, which could otherwise lead to unwanted well contamination or spillage. Additionally, foil seals protect light-sensitive reagents. \ \ Pro-tip: If you are going to store sealed plates use a [roller tool ](https://www.thomassci.com/Molecular-Diagnostics/Automation/Plates/Sealing/_/Sealing-Film-Accessories)to really make sure the edges are sealed tightly or else you may get evaporation!
{% hint style="info" %} If you have any questions, comments, or concerns with your Parhelia products please email [info@parheliabio.com](mailto:undefined) {% endhint %} # S12 Assembly and Sample Prep How to put together your Omni-Stainer™ S12 system and prepare samples for staining ## Step 1 - Create Humid Environment: add 30 mL deionized water to the Omni-Stainer™ S12 base

This helps to maintain a humid environment and prevents evaporation

## Step 2 - Construct S-type Flow Cell: mounting a Cover Pad on a slide with a sample. Adhesive spacers on the plastic Cover Pads ensure optimal flow cell thickness. The chosen adhesive allows the Cover Pad to slide when wet but holds it in place to prevent lateral staining solution bleed. **To place the coverpad** First remove the coverpad from the plastic film. Then, pre-wet both the coverpad and the slide surface. Then slide on the coverpad over the sample such that the "flow cell" is evenly filled with the liquid (PBS or hydration buffer) without any bubbles (see videos below). There are multiple methods to build the flow cell "sandwich," aiming to ensure the Cover Pad sits firmly on the slide, and the internal "capillary chamber" contains buffer without any air bubbles. ### The "Yury Method" {% embed url="" %} Yury Method, slightly more finesse than the "Easy Method" but fits into tissue prep workflows and saves labware. {% endembed %} ### The "Easy Method" {% embed url="" %} Easy Method, \*Note: Cover Pads now have peelable plastic film on both sides, rather than peel tabs. {% endembed %} Easy Method" consists of sliding on the Cover Pad under the layer of PBS or any such compatible buffer inside a suitable reservoir such as a pipette tip box lid. ### The "Big Drop Method" As confidence and experience are built, constructing an S-type flow cell can also be achieved by placing a big drop (250ul) of compatible buffer on top of the slide and then sliding the coverpad over the drop to avoid bubbles. {% hint style="info" %} If bubbles are entrapped – sliding the coverpad such that the bubble is released and then sliding it back after adding a bit more of the liquid allows to "expel" the bubbles {% endhint %}
Preparing different sample types * **FFPE sections:** a sample needs to be baked, deparaffinized with Xylenes or a suitable alternative, and re-hydrated. If HIER is needed, at this time we recommend it to be. It is possible to do HIER directly in Parhelia Omni-stainer using the Thermal Sheath, but that functionality is experimental and hasn’t been fully tested. Please reach out to us directly at if you would like to try running HIER with Parhelia Omni-stainer. * **Fresh frozen sections:** sample needs to be fixed and re-hydrated * **Cell culture/cell spreads:** samples needs to be fixed and re-hydrated
## Step 3 - Place flow cell into the shelf, and insert shelf into base

Place flow cell into shelf

Place shelf into base

## Step 4 - Adhere moisture barrier paper to the lid's bottom, wet it with 10 mL of deionized water

Make sure the holes are aligned if using automation lid

Humidity barrier placement for manual lid

## Step 5 - Put the lid on the base, matching slanted corners

Align the slanted and rounded corners of the lid and base.

## Full Assembly

Insert sample, then put on lid (automation lid shown)

S12 Automation Specs ![](/files/cF110aThRFMKsbFWbeyN)
S12 Manual Specs ![](/files/vX3BrLGWesj1xhTm6SQP)
## Using a Thermal Sheath? {% content-ref url="/pages/kLgIdhM9OVgNHKqLJidY" %} [Thermal Sheath & Temperature-controlled incubations](/product-guides/automation-opentrons-ot-2-setup/thermal-sheath-and-temperature-controlled-incubations) {% endcontent-ref %} {% hint style="success" %} Done! You're now ready to choose Manual or Automated mode and launch the demo! {% endhint %} # C12 Assembly and Sample Prep How to put together your Omni-Stainer™ C12 system and prepare samples for staining ## Step 1 - Create Humid Environment: add 30 mL deionized water to the Omni-Stainer™ C12 base

This helps to maintain a humid environment and prevents evaporation

## Step 2 - Construct Flow Cell and Place in Base You can create a flow cell in two methods: 1. \[Shown in video below] Add \~100 ul of PBS on the Support Pad, and then place the coverslip (sample side down) onto the support pad. Adjust the coverslip ensuring it aligns with the lower boundary of the Support Pad. If there are air bubbles, sliding the coverslip such that bubble is released and then sliding it back after adding a bit more of the liquid allows to "expel" the bubbles 2. Align the coverslip and Support Pad "underwater" within a PBS pool, this technique that demands less skill to avoid air bubbles - however, the delicacy and clearness of coverslips occasionally make this less suitable for the C-style flow cell than for the S-style. {% embed url="" %} A flow cell can be made with a drop of PBS (as in this video) or by constructing a flow cell within a reservoir of PBS {% endembed %}
Preparing C type flow cell Both the S (slide) and the C (coverslip) Omni-Stainers utilize "capillary liquid exchange " concept which is based on creating a thin layer of staining solution evenly covering the sample. The space where the sample is facing the exchangeable reactive staining solutions is called a "flow cell". To create the flow cell in C type Omni-Stainer the coverslip with the sample facing down needs to placed onto a Support Pad. This can be achieved in a number of ways – the easiest one by doing it under the layer of PBS or any such compatible buffer inside a tip box lid or a beaker. As confidence and experience are built this can also be done by placing a drop of compatible buffer on top of the Support Pad and then covering the drop with a coverslip such that bubbles are avoided. In the case bubbles are encountered – sliding the coverslip such that bubble is released and then sliding it back after adding a bit more of the liquid allows to "expel" the bubbles
Preparing different sample-types * **FFPE sections:** a sample needs to be baked, deparaffinized with Xylenes or a suitable alternative and re-hydrated. If HIER is needed, at this time we recommend it to be. It is possible to do HIER directly in Parhelia Omni-stainer using the Thermal Sheath, but that functionality is experimental and hasn’t been fully tested. Please reach out to us directly at if you would like to try running HIER with Parhelia Omni-stainer. * **Fresh frozen sections:** sample needs to be fixed and re-hydrated * **Cell culture/cell spreads:** samples needs to be fixed and re-hydrated
## Step 3 - Confirm Capillary Connection It is important to verify that the coverslip aligns perfectly with the lower boundary of the Support Pad and makes contact with the wicking pillar. Confirm the existence of a capillary connection between the staining chamber and the wicking pillar, which essentially entails a liquid droplet linking the two components.\ \ If that’s not the case, dispense 100ul of PBS onto the contact point between the wicking pillar and the support tile (see diagram below).
## Step 4 - Adhere moisture barrier paper to the lid's bottom, wet it with 10 mL of deionized water

Make sure the holes are aligned if using automation lid

Humidity barrier placement for manual lid

## Step 5 - Put the lid on the base, matching slanted corners

Align cut diagonal corner of lid and base

## Full Assembly

Insert sample, then put on lid (automation lid shown)

Complete Assembly (manual lid, 12 support pads)

C12 Automation Specs ![](/files/yThPVkA3PaW8WqOkOu1T)
C12 Manual Specs ![](/files/xQ9xCWvB2ekcRalw013j)
## Using a Thermal Sheath? {% content-ref url="/pages/kLgIdhM9OVgNHKqLJidY" %} [Thermal Sheath & Temperature-controlled incubations](/product-guides/automation-opentrons-ot-2-setup/thermal-sheath-and-temperature-controlled-incubations) {% endcontent-ref %} {% hint style="success" %} Done! You're now ready to choose Manual or Automated mode and launch the demo! {% endhint %} # Sample Mounting Instructions To ensure bubble-free operation, use these directions for S-type and C-type flow cell construction / sample mounting. \*This information is also in [**S12** **Assembly and Sample Prep**](/product-guides/s12-assembly-and-sample-prep) and [**C12 Assembly and Sample Prep**](/product-guides/c12-assembly-and-sample-prep)**.** ## Sample Mounting for S12 Parhelia Omni-Stainer™ Two adhesive spacers running along the edges of the single-use plastic Cover Pads are calibrated to maintain the optimal thickness of the flow cell. The adhesive is carefully chosen in such a way that when wet the Cover Pad can slide along the surface of the slide and yet when slightly pushed against the slide the adhesive holds the position of the coverpad and prevents the lateral bleed of the staining solutions. ## How to construct S-type Flow Cell: mounting a Cover Pad on a slide with a sample. ### Placing a Cover Pad First remove the Cover Pad from the plastic film. Then, pre-wet both the coverpad and the slide surface. Then slide on the coverpad over the sample such that the "flow cell" is evenly filled with the liquid (PBS or hydration buffer) without any bubbles (see videos below). There are multiple methods to build the flow cell "sandwich," aiming to ensure the Cover Pad sits firmly on the slide, and the internal "capillary chamber" contains buffer without any air bubbles. ### The "Simple Method" "Simple Method" consists of sliding on the Cover Pad under the layer of PBS or any such compatible buffer inside a suitable reservoir such as a pipette tip box lid. {% embed url="" %} ### The "Yury Method" The Yury Method can be used with classic or glass Cover Pads, but is especially useful if using Glass Cover Pads which have lower tack. {% embed url="" %} S-type flow cell preparation for Parhelia Omni-Stainer™ S12 Module Yury Method uses slightly more finesse than the "Simple Method" but fits into some tissue prep workflows more easily and is especially useful if using glass Cover Pads. ### Plastic "Classic" Cover Pad (deprecated) methods Just in case anyone has these out there. We only sell the glass now, since the plastic can generate gas bubbles in heating. {% embed url="" %} As confidence and experience are built, constructing an S-type flow cell can also be achieved by placing a big drop (250-500ul) of compatible buffer on top of the slide and then sliding the coverpad over the drop to avoid bubbles. {% embed url="" %} {% hint style="info" %} If bubbles are entrapped – sliding the coverpad such that the bubble is released and then sliding it back after adding a bit more of the liquid allows to "expel" the bubbles {% endhint %}
Preparing different sample types * **FFPE sections:** a sample needs to be baked, deparaffinized with Xylenes or a suitable alternative, and re-hydrated. It is also possible to do HIER directly in Parhelia Omni-stainer using the Thermal Sheath and temperature module. * **Fresh frozen sections:** sample needs to be fixed and re-hydrated * **Cell culture/cell spreads:** samples needs to be fixed and re-hydrated
## Additional Flow Cell Prep Videos [Yury Method with Classic Cover Pad \[Video Link\]](https://vimeo.com/852759019) [Big Drop Method with Glass Cover Pad \[Video Link\]](https://vimeo.com/852759019) {% embed url="" %} Yury Method, as explained by Yury - Glass Cover Pad {% endembed %} ## How to construct C-type Flow Cell: mounting a cover tile on a Support Pad | C12 Parhelia Omni-Stainer™ {% embed url="" %} # Manual Operation and "Blue Dye Demo" Manual Protocol The Manual Product Bundles and the Automated Product Bundles can both be operated manually with a P200 micropipette. ## Follow steps 1-3 Assembly and Sample Prep guide for your Omni-Stainer type: {% content-ref url="/pages/NmuN25IFo3T1ISCeUP2R" %} [S12 Assembly and Sample Prep](/product-guides/s12-assembly-and-sample-prep) {% endcontent-ref %} {% content-ref url="/pages/1H3JOB80iwMXtLYKepXk" %} [C12 Assembly and Sample Prep](/product-guides/c12-assembly-and-sample-prep) {% endcontent-ref %} {% hint style="info" %} Remember to add 20-30 ml of distilled water to the Omni-Stainer base. {% endhint %} ## Now, exchange the solutions in the flow cell. (like videos) {% embed url="" %} Omni-Stainer™ S12, Manual Blue Dye Demo Video {% endembed %} {% embed url="" %} Omni-Stainer™ C12, Manual Blue Dye Demo Video {% endembed %} To exchange the current solution in the flow cell with a new solution – pipette the new solution onto the slide or coverslip support pad surface slightly (\~2mm) above the interface of the flow cell with air. The new solution should be applied with a speed slow enough that it does not overflow over the surface of the coverslip or Support Pad. Ideally, the end of the pipette tip should be positioned close enough to the surface such that the solution immediately transitions from the tip to the surface avoiding the formation of drops. Sometimes especially before sufficient proficiency in handling the Omni-Stainers is acquired slight occasional overflow over the surface of the flow cell may occur. This does not constitute any danger and does not impact the final staining quality The laminarity of the solution flow in the flow cell starts as solution spreading across the upper edge of the Cover Pad or coverslip. The faster the solution flows the less is the diffusion at the exchange front and hence the less is the volume that is needed for complete solution exchange. Currently, the volume needed to replace the solution under the S-type Cover Pad is 150 μl and inside the C-type Support Pad is 100 μl. {% hint style="info" %} Our engaging virtual demonstration serves as an excellent introductory guide for understanding the steps in operating the Parhelia Bio Omni-Stainer C12 and S12, encompassing both manual and automated usage, along with thermal sheath and thermal modules. [**View the virtual demo here!**](https://parheliabio.com/view-virtual-demo/?utm_source=gitbook\&utm_medium=gitbook) {% endhint %} ## Manual Blue Dye Demo This is a very simple demo protocol that helps you understand how capillary gap exchange works inside Parhelia Omni-stainer and calibrate volumes that are needed for the gap exchange. #### Manual Blue Dye Protocol * Prepare two \[Eppendorf] tubes and fill one with PBS and the other with Blue Dye (1.0 mL each). * Utilizing a P200 pipette, gradually dispense 110 μl of Blue Dye onto the upper curved edge of the S12 Cover Pads (for C12 Support Pads, dispense 60ul of the solution onto the ramp). Observe attentively as the Blue Dye is drawn into the chamber, ensuring complete displacement of the clear liquid previously present in the staining chamber, which should drip down the wicking pillar. Subsequently, cleanse the staining chamber by dispensing 150 μl of the clear buffer, ensuring complete displacement of the blue dye. * Refer to the provided videos for visual guidance. It is normal to have very small pockets of carryover buffer in the lower corners of the staining chamber, which should not affect the quality of staining since there is usually no tissue present in those areas. * However, if you desire a more thorough exchange, you can increase the wash volume as needed. If the flow appears unusually slow and the exchange seems incomplete and uneven: * Check for bubbles in the chamber. If bubbles are present, slide the sample up and down to release the bubble. If that does not resolve the issue, remount the sample. * Verify that the sample is correctly positioned within the staining chamber. # Automation (Opentrons OT-2) Setup This page goes through setting up a new Opentrons OT-2, most of the content is in links to Opentrons website and Opentrons videos. {% hint style="info" %} This is for automation bundles only, click here for manual operation:[Manual Operation and "Blue Dye Demo" Manual Protocol](/product-guides/manual-operation-and-blue-dye-demo-manual-protocol) {% endhint %} {% hint style="info" %} #### If you want to get a head start, download our GitHub archive from [here](https://github.com/parheliabio/ot2-scripts/archive/refs/heads/main.zip)! {% endhint %} ## Opentrons OT-2 Robot and Software setup {% embed url="" %} ^ Click here ^ For Opentrons Set up {% endembed %} The Opentrons Help Center at is a fantastic resource that has s[tep-by-step guides](https://support.opentrons.com/s/article/Get-started-Unbox-the-OT-2) as well as [**video guides**](https://support.opentrons.com/s/article/Get-started-Unbox-the-OT-2) **(which we strongly recommend) like this video series below** that takes you from box to protocol. {% embed url="" %} ^ This playlist covers ^ 1. [Unboxing the OT-2](https://youtu.be/nvjNHod-2hU) 2. [Unlocking the OT-2](https://youtu.be/c2fnzlFfCSE) 3. [Setting up the Opentrons App](https://youtu.be/sqkvy8xdPVw) 4. [Attaching Pipettes to the OT-2](https://youtu.be/xmCItodWx2o) 5. [Deck Calibration on the OT-2](https://youtu.be/bKYmFyJACaU) 6. [Tip Length & Pipette Offset Calibration on the OT-2 ](https://youtu.be/7mMtvdcV-dM) 7. [Running a Protocol on the OT-2](https://youtu.be/hiN0EOW3vHM) {% hint style="info" %} The Deck Calibration process in video 5 of jogging the pipette tip in the XYZ directions into the center of the engraved "+" symbol on the Opentrons deck is very similar to the labware calibration for the Omni-Stainer™ which is performed using the embossed "+" symbol on the C12 or S12 Omni-Stainer™ lid.\ [As in this video ](https://vimeo.com/779408843) {% endhint %} {% hint style="success" %} Our engaging virtual demonstration serves as an excellent introductory guide for understanding the steps in operating the Parhelia Bio Omni-Stainer C12 and S12, encompassing both manual and automated usage, along with thermal sheath and thermal modules. [**View the virtual demo here!**](https://parheliabio.com/view-virtual-demo/?utm_source=gitbook\&utm_medium=gitbook) {% endhint %} ## Checklist * [ ] Set up Opentrons OT-2 robot. [This set-up guide is a great reference](https://support.opentrons.com/s/ot2-get-started), and our friends at Opentrons are happy to assist you with this if you have any [questions](https://support.opentrons.com/s/information-about-opentrons-support). * [ ] [Download Opentrons App](https://opentrons.com/ot-app/) * [ ] [Download Parhelia Bio's base OT-2 scripts and labware definitions from GitHub by clicking here](https://github.com/parheliabio/ot2-scripts/archive/refs/heads/main.zip) * [ ] If you would prefer to visit the GitHub and download individual files our GitHub is at * [ ] Download customized or stock Omni-stainer script from [Stainworks](https://3qcwn6iy48.execute-api.us-east-1.amazonaws.com/) * [ ] Load protocols and labware definitions into Opentrons software * [ ] Follow protocol-specific instructions, for preparing and loading your labware and doing protocol setup. We strongly recommend starting with the Blue Dye Demo protocol to get comfortable using the device before conducting actual experiments.

Example of staining protocol loaded into Opentrons

# Omni-Stainer Automation Overview Roadmap for Getting Set up with the Omni-Stainer in an Opentrons OT-2 * [ ] Set up Opentrons OT-2 robot. [This set-up guide is a great reference](https://support.opentrons.com/s/ot2-get-started), and our friends at Opentrons are happy to assist you with this if you have any [questions](https://support.opentrons.com/s/information-about-opentrons-support). * [ ] [Download Opentrons App](https://opentrons.com/ot-app/) * [ ] [Download Parhelia Bio's base OT-2 scripts and labware definitions from GitHub by clicking here](https://github.com/parheliabio/ot2-scripts/archive/refs/heads/main.zip) * [ ] If you would prefer to visit the GitHub and download individual files our GitHub is at * [ ] Download customized or stock Omni-stainer code from [Stainworks](https://3qcwn6iy48.execute-api.us-east-1.amazonaws.com/) * [ ] Load protocols and labware definitions into Opentrons software * [ ] Follow protocol-specific instructions, for preparing and loading your labware and doing protocol setup. We strongly recommend starting with the Blue Dye Demo protocol to get comfortable using the device before conducting actual experiments.

Example of staining protocol loaded into Opentrons

## We suggest starting with the "Blue Dye Demo" Automated Protocol before exploring other protocols. This demo's steps are crafted to generally adapt to various Parhelia protocols. Find the protocol here: {% content-ref url="/pages/C6Pql6XPM8NNOELpQPXx" %} ["Blue Dye Demo" Automated Protocol](/product-guides/blue-dye-demo-automated-protocol) {% endcontent-ref %} Our engaging virtual demonstration serves as an excellent introductory guide for understanding the steps in operating the Parhelia Bio Omni-Stainer C12 and S12, encompassing both manual and automated usage, along with thermal sheath and thermal modules. [**View the virtual demo here!**](https://parheliabio.com/view-virtual-demo/?utm_source=gitbook\&utm_medium=gitbook) ## Step 1 - Place the Omni-Stainer, reagent reservoirs, pipette tip rack, and antibody plate into the dedicated slots on the deck of the liquid handler. When inserting the labware (plates, reservoirs, Omni-Stainers) into the slot of the liquid handler deck it is important to ensure the complete in-flush alignment of the labware bottom with the surface of the deck. Lack of such alignment may result in tips bending while hitting the bottom of the labware wells. SBS labware should click in or rest fully in a deck location, a common error is one of the back corners not being fully inserted and level with the rest of the labware. ## Step 2 - Create a running protocol using the Staining Works Software The Python files with staining protocols for an individual experiment can be easily configured using the Staining Works Software. For auditing and accountability we recommend storing the individual day–specific protocols and assigning them a name consisting of the operator name, date, and the purpose of the experiment. For more information go to our StainWorks documentation page: {% content-ref url="/pages/MkHx9WbtUtDL8OD8LfBh" %} [StainWorks](/product-guides/automation-opentrons-ot-2-setup/stainworks) {% endcontent-ref %} You may see a protocol analysis failure in the Opentrons App if the necessary labware definitions have not been uploaded. To learn more see this documentation page: {% content-ref url="/pages/nKQMxb6PDGLrLArrVpt2" %} [Importing labware definitions](/product-guides/automation-opentrons-ot-2-setup/importing-labware-definitions) {% endcontent-ref %} ## Step 3 - Start the experiment. Calibration step. ## Step 4 - Start the experiment. Initiate the protocol run. # Importing labware definitions Custom labware definitions have been meticulously created for Parhelia Omni-stainer instruments and reservoirs, these files are essential for running Parhelia Bio protocols. Labware definitions are specialized JSON format files that contain essential information about the labware's geometry, such as the number of wells, XY positioning, and well depth. These files are crucial for enabling the Opentron OT-2 robot to accurately access the samples. To facilitate this process, we have developed custom labware definitions specifically for Parhelia Omni-stainer instruments and reservoirs. To get started, simply **download our GitHub** [**here**](https://github.com/parheliabio/ot2-scripts/archive/refs/heads/main.zip), unzip the files, and **follow** [**this guide**](https://support.opentrons.com/s/article/Using-labware-in-your-protocols) to import the JSON files from the labware folder into the Opentrons app. Click below if you wish to download just the labware definition files: {% file src="/files/A11WIT3tLhlwuXRO7vDS" %} Opentrons Labware Definitions .JSON Files {% endfile %} You may have to upload custom labware definitions one at a time. ## Troubleshooting ### Protocol analysis failure In this example the protocol has already been configured and downloaded from StainWorks, and uploaded to Opentrons App, this process is covered in these two document pages. {% content-ref url="/pages/C6Pql6XPM8NNOELpQPXx" %} ["Blue Dye Demo" Automated Protocol](/product-guides/blue-dye-demo-automated-protocol) {% endcontent-ref %} {% content-ref url="/pages/xzxNw62cWQpoXqHIkjIj" %} [Automation (Opentrons OT-2) Setup](/product-guides/automation-opentrons-ot-2-setup) {% endcontent-ref %} If a protocol is loaded but the labware definition is missing the protocol will show up looking like:
And the error details will look like:

Missing labware definition error details

If this happens, import the missing labware definitions, and then click "Reanalyze protocol" in the Protocols tab. If the labware definitions are not missing, and you click on the protocol you should see something like this:

Example of protocol with correct labware definitions, the lack of a blue check next to the labware is normal and does not mean there is any missing labware definitions.

# Opentrons App v7 Updated Labware Definitions If running Opentrons app v7.0 or above, the "v7" version of the labware should be used to avoid automation errors. We have an important update regarding the recent Opentrons app v7 software. An issue with the latest update may cause labware definitions to behave unexpectedly. We recommend updating your Labware definitions with those in the zip below: {% file src="/files/TUQE434MZrMuJNdoLaWJ" %} Updated labware for Opentrons App v7 and above {% endfile %} [ (or grab the "v7" files from our GitHub page)](https://github.com/parheliabio/ot2-scripts/tree/main/labware) {% embed url="" %} How to replace an existing labware definition {% endembed %} Fresh protocols from Parhelia Stainworks will automatically load the new labware definitions, but to update previously run protocols, you will need to click "Reanalyze". {% embed url="" %} How to reanalyze previously run protocols to update labware def {% endembed %} {% hint style="info" %} Make sure to calibrate the labware definition offset the first time you run a new labware or use it in a deck position for the first time. {% endhint %} ## Labware Offset Calibration The updated labware do not require the manual 2 mm up offset like the prior labware. Calibrate to the calibration points as described on the Opentrons App {% embed url="" %} Labware Offset Calibration Walk Tutorial {% endembed %}

Calibration point for S12 with thermal sheath on thermal module is the top of the NorthWest screw

# StainWorks StainWorks allows for simple no-code customization of staining protocols. ## StainWorks Since many researchers are not familiar with with Python, we at Parhelia created StainWorks: a simple yet powerful web app that provides two essential features: #### Templating The ability to configure Python protocols without having to dig into the Python code. Instead, the relevant run parameters (i.e., number of samples, wash volumes, incubation times). #### Global Library A curated collection of Python scripts that implement various assays (CODEX, IHC, H\&E etc). These protocols have been created, validated and maintained by the Parhelia team. ## Labware layouts Each template in StainWorks comes with a unique “Labware Layout” spreadsheet that is attached to the template. Labware Layout contains instructions on how to correctly fill reservoirs with reagents and place them onto the deck of an OT-2 robot for a particular protocol run. These spreadsheets have modifiable input fields (i.e., Omni-Stainer type, Number of samples) that generate a lot of information for how to set up the experiment. {% hint style="info" %} Accessing StainWorks requires having a user account. If you purchased an S12 or C12 automation bundle, your StainWorks account will be set up automatically and a 90-day free trial of StainWorks is included with your purchase. You will receive a separate email from with your account credentials. You can learn more about exactly how to use StainWorks in the “Blue Dye Demo” tutorial below. {% endhint %}

StainWorks UI showing customization fields to configure unique protocols

## To continue with StainWorks-based Automation Setup, click here --> [Labware Layouts](/product-guides/automation-opentrons-ot-2-setup/labware-layouts)

Some auto-staining procedures we have developed and validated for the Omni-Stainer

{% hint style="info" %} Also, if you want to try a staining procedure we have not already made available, we do offer [custom protocol development as a service](https://parheliabio.com/product/custom-protocol-development/?utm_source=gitbook\&utm_medium=gitbook\&utm_content=protocol_customization). {% endhint %} Some users may like to develop custom Omni-Stainer protocols on their own. Below is some information to assist in the user development of custom OT-2 protocols with the Omni-Stainer. ### User Python Protocol Programming Outside StainWorks Opentrons protocols are written using the Python programming language and make use of Opentrons’ Python API. This innovative feature of the OT-2 makes good use of Python’s combination of simplicity and expressive power, so you can easily program arbitrarily complex protocols even as a beginner. Follow this [comprehensive guide on using Python for OT-2 with Protocols](https://docs.opentrons.com/v2/writing.html) to learn more #### Below are our global functions used throughout our protocols, it is available along with our latest public code updates at: {% code title="global\_functions.py" overflow="wrap" lineNumbers="true" %} ```python //## VERAO GLOBAL ## Copyright Parhelia Biosciences Corporation 2022-2023 ### GLOBAL FUNCTIONS - AUTO-GENERATED - DO NOT MODIFY ### from opentrons import protocol_api import json class Object: pass ####################GENERAL SETUP################################ stats = Object() stats.volume = 0 debug = False ####################FIXED RUN PARAMETERS######################### default_flow_rate = 50 well_flow_rate = 5 sample_flow_rate = 0.2 extra_bottom_gap=0 def washSamples(pipette, sourceSolutionWell, samples, volume, num_repeats=1, height_offset=0, keep_tip=False): try: iter(samples) except TypeError: samples = [samples] print('Samples are:') print(samples) if not pipette.has_tip: pipette.pick_up_tip() height_offset += extra_bottom_gap for i in range(0, num_repeats): for s in samples: print(s) print("Washing sample:" + str(s)) pipette.aspirate(volume, sourceSolutionWell.bottom(height_offset), rate=well_flow_rate) pipette.dispense(volume, s.bottom(height_offset), rate=sample_flow_rate) stats.volume += volume if not keep_tip: pipette.drop_tip() def puncture_wells(pipette, wells, height_offset=0, keep_tip=False): try: iter(wells) except TypeError: wells = [wells] for well in wells: washSamples(pipette, well, well, 1, 1, height_offset, keep_tip=True) if not keep_tip: pipette.drop_tip() def dilute_and_apply_fixative(pipette, sourceSolutionWell, dilutant_buffer_well, samples, volume, height_offset=0, keep_tip=False): if not pipette.has_tip: pipette.pick_up_tip() # Diluting fixative: pipette.aspirate(volume, dilutant_buffer_well, rate=well_flow_rate) pipette.dispense(volume, sourceSolutionWell, rate=well_flow_rate) for iterator in range(0, 3): pipette.aspirate(volume, sourceSolutionWell, rate=well_flow_rate) pipette.dispense(volume, sourceSolutionWell, rate=well_flow_rate) washSamples(pipette, sourceSolutionWell, samples, volume, 1, height_offset, keep_tip=keep_tip) def getOmnistainerWellsList(omnistainer, num_samples): sample_chambers = [] if (len(omnistainer.wells_by_name()) < num_samples): raise Exception("number of wells in the Omni-Stainer less than num_samples") wellslist = list(omnistainer.wells_by_name().keys()) wellslist = wellslist[1:num_samples + 1] for well in wellslist: sample_chambers.append(omnistainer.wells_by_name()[well]) print("omnistainer.wells_by_name are:") print(omnistainer.wells_by_name()) print("sample_chambers are:") print(sample_chambers) return sample_chambers def mix(pipette, sourceSolutionWell, volume, num_repeats): if not pipette.has_tip: pipette.pick_up_tip() for i in range(0, num_repeats): pipette.aspirate(volume, sourceSolutionWell, rate=2) pipette.dispense(volume, sourceSolutionWell, rate=2) pipette.drop_tip() def openShutter(protocol, pipette, covered_lbwr, keep_tip=False): if not pipette.has_tip: pipette.pick_up_tip() pipette.move_to(covered_lbwr.wells()[len(covered_lbwr.wells()) - 2].bottom(0)) pipette.move_to(covered_lbwr.wells()[len(covered_lbwr.wells()) - 1].bottom(0), force_direct=True) protocol.delay(seconds=1) if not keep_tip: pipette.drop_tip() def closeShutter(protocol, pipette, covered_lbwr, keep_tip=False): if not pipette.has_tip: pipette.pick_up_tip() pipette.move_to(covered_lbwr.wells()[len(covered_lbwr.wells()) - 2].bottom(0)) pipette.move_to(covered_lbwr.wells()[len(covered_lbwr.wells()) - 3].bottom(0), force_direct=True) protocol.delay(seconds=1) if not keep_tip: pipette.drop_tip() ### END VERAO GLOBAL ``` {% endcode %} # Labware Layouts Labware Layout files are .xlsx files that differ in complexity depending on the complexity of the staining protocol. ## Deck Layout The first thing labware layouts do is indicate what labware and what instruments go where. This information is also available in the Opentrons App. ## Reagent Layout The crucial aspect of labware layout files is discerning which reagents, volumes, and concentrations go where. These files contain input fields to define experiment dimensions, automatically populating various fields. StainWorks also does these setups, the difference being StainWorks outputs robot-friendly code, while labware layouts produce human-readable spreadsheets. Below is a screenshot illustrating the CODEX protocol, among our more intricate staining protocols:

Drop down selection fields allow for customization

# Thermal Sheath & Temperature-controlled incubations This page is under construction, please reach out to our team for help with the thermal sheath and temperature modules. {% hint style="warning" %} :construction: This page is under construction :construction: {% endhint %} Congrats on choosing the Omni-Stainer™ thermal sheath S12 / C12!\ \ The Omni-Stainer™ is made of heat-conductive aluminum and can be used in conjunction with a temperature control module, allowing the temperature to be programmatically controlled between -20° to 99.9°C. This enables a variety of protocols, such as FFPE Antigen Retrieval, smRNA-FISH, or CycIF to be performed directly inside the Omni-Stainer™ device.\ \ To run thermal incubations there are 3 main things you need 1. A thermal module. We recommend QInstruments ColdPlate 2. A temperature-controlled program 3. A thermal sheath and Opentrons robot

Omni-Stainer going onto thermal module and covered with thermal sheath

### [QInstruments ColdPlate Product Page](https://www.qinstruments.com/automation/coldplate/) ## Temperature-controlled protocols * Assemble Omni-Stainer as in the Assembly and Sample Prep protocols, make sure there is sufficient water in the base and the humidity barrier paper. * [S12 Assembly and Sample Prep](/product-guides/s12-assembly-and-sample-prep) * [C12 Assembly and Sample Prep](/product-guides/c12-assembly-and-sample-prep) * Place thermal sheet on top of the Omni-Stainer * Set up the temperature module on the deck of the QInstruments ColdPlate manual. * When running the protocols, make sure to select “omni\_stainer\_###\_with\_thermosheath”, where ### is the instrument model (i.e. S12\_slides or C12\_cslps) * During the calibration step, **calibrate the pipette tip to the calibration point on top of the thermal sheath**
{% hint style="danger" %} When creating your own protocol, ensure that you utilize the`openShutter(...),` and `closeShutter(...)` functions from the protocol header. **Neglecting to do so may result in damage to your pipette, for which we cannot be held responsible**. {% endhint %} ## Temperature Modules At Parhelia , we value efficient, precise equipment. That's why for the Opentrons OT-2 to effectively utilize the Omni-Stainer **S12 or C12** with a thermal sheath, we recommend the QInstruments thermal module. The current Opentrons thermal module is slightly too tall for the Omni-Stainer with thermal sheath, making QInstruments the ideal alternative. Their ColdPlate ensures unrivaled heating and cooling precision for demanding robotic applications. \ [QInstruments ColdPlate Product Page](https://www.qinstruments.com/automation/coldplate/) ## QInstruments ColdPlate Compact, lightweight, effective, and silent, it provides heating and cooling combined in a single unit. **Fully adjustable from -20° to 99.9°C**, its optimized thermo adapters and premium aluminium housing ensure excellent temperature control. Opentrons support remains available for assistance, despite this being a third-party module. With an easy setup and an extended warranty, the QInstruments module is a valuable addition to any laboratory setup. {% hint style="info" %} If you have any questions, comments, or concerns please email [info@parheliabio.com](mailto:undefined) {% endhint %} # "Blue Dye Demo" Automated Protocol This demo protocol demonstrates the basics of automated capillary gap staining liquid exchange with an C12/S12 Omni-Stainer™ ## Blue Dye Demo Automated Protocol * [ ] **Step 1:** Open [StainWorks](https://3qcwn6iy48.execute-api.us-east-1.amazonaws.com/?utm_source=gitbook\&utm_medium=gitbook\&utm_content=stainworks)

Colored arrows indicate the click locations for corresponding colored steps

* [ ] **Step 2:** Go to “Global Library”, locate “Blue Dye Demo” protocol and copy it to your Templates * [ ] **Step 3:** Go to “Templates”, locate “Blue dye demo…” protocol Download the ‘labware layout\_Blue\_dye\_demo\_...xlsx’ file and follow the instructions in the file for filling the reservoir. Labware layout files can contain a lot of useful information for more complex protocols. * [ ] **Step 4:** * [ ] In StainWorks, in Templates, press “modify protocol”\ <- Modify button circled * [ ] In the dialogue that pops up, select the type of your Omni-Stainer (C12 or S12), keep all other values default. * [ ] Press “Save and Download” * [ ] **Step 5:** Upload the .py file that you just downloaded into the Opentrons app.\ {% embed url="" %} This video is set to start at 1:38 which shows uploading a protocol into Opentrons, and tip calibration for a general protocol \ (see step 8 for Omni-Stainer specific calibration) {% endembed %} {% hint style="info" %} If you did not upload the custom labware to Opentrons, you will see a protocol analysis error. The labware definitions needed for the demo are below for your convienance. \ The labware may need to be imported one at a time.\ Check out the [Importing labware definitions](/product-guides/automation-opentrons-ot-2-setup/importing-labware-definitions) guide for more info! {% endhint %} {% file src="/files/3FEk20UExu1v9zFDNd15" %} Omni-Stainer S12 labware definition {% endfile %} {% file src="/files/zRCsulhiHO25uRnUY75M" %} 12-trough (AKA 12-well or 12-channel) labware definition {% endfile %} {% file src="/files/qSBRLnCNhVVidhiUIzHc" %} Omni-Stainer C12 labware definition {% endfile %} {% hint style="info" %} Remember to add 20-30 ml of distilled water to the Omni-Stainer base. {% endhint %} * [ ] **Step 6:** Place the labwares on the deck according to the Deck View in the Opentrons app (the same information is also in the labware layout file), making sure that the recessed corner of the Omni-Stainer is in the northwest (top-left) position, also refer to[ this video](https://www.google.com/url?q=https://vimeo.com/781366854\&sa=D\&source=editors\&ust=1684536753676829\&usg=AOvVaw1nmhX8wFYZlNBvxB2nGHAk).

If all the files are uploaded correctly you should see this
(or S12 replaced with C12 if using C12)

{% hint style="info" %} Make sure all labware (plates, reservoirs, Omni-Stainers) is flat on the liquid handler deck to stop tips from bending. SBS labware should fully click into or sit in the deck slot. Avoid usual mistake of back corners not fully in or level. {% endhint %} * [ ] **Step 7:** Prepare a blank sample and place it into position 1 (the one closest to the recessed corner) of the Parhelia Omni-Stainer * S12 Omni-Stainer: Take a blank microscopy slide, wipe it clean and dry and mount it with a Cover Pad according to the directions in [**S12 Assembly and Sample Prep**](/product-guides/s12-assembly-and-sample-prep) section. * C12 Omni-Stainer: Take a blank coverslip, wipe it clean and dry and mount it on a Support Pad according to the directions in [**C12 Assembly and Sample Prep**](/product-guides/c12-assembly-and-sample-prep) section. * [ ] **Step 8:** Close the lid of the Omni-Stainer. Visually locate the calibration mark, which looks like + sign embossed on the lid (note that for C12 and S12, the marks are in different locations). Proceed to run the protocol calibration with the lid closed. \* \[ ]

Omni-Stainer labware calibration window in Opentrons app, use adjustment controls to get pipette tip on the center of the "+" symbol

\* \[ ]

Images of proper tip alignment, shown for the S12 automation lid

\* \[ ]

The tip calibration location is Northwest for the S12 and Southwest for the C12

* [ ] [**Tip calibration for the various Parhelia labware and the Omni-Stainer can also be seen in this video.**](https://vimeo.com/779408843) * [ ] **Step 9:** If calibration looks good, proceed with the protocol run. * [ ] **Step 10:** Remove the lid and run the protocol to observe the exchange. It should look like the videos below. * [ ] **Step 11 (optional):** **How to re-run the protocol (without re-calibrating):** Just clicking “Run again”, then “Start Run” will not apply your saved calibration data and the pipetting locations could be off. To simply re-run a previously loaded protocol, select “Protocols” on the left side, choose the protocol, then select Run protocol, choose the OT-2 robot and select “Proceed to Setup”. On the Setup tab click open "STEP 1 Robot Calibration", click Proceed to labware setup. A pop-up window will appear will the calibration data, click "**Apply stored data"**, click “Proceed to Run”. Click “Start run” ## Videos of what the Blue Dye Demo looks like {% embed url="" %} Omni-Stainer™ S12 running Dye Demo (sped up) {% endembed %} {% embed url="" %} Omni-Stainer™ C12 running Dye Demo (sped up) {% endembed %} note: The videos above show violet dye in well 1 and clear in well 2, while this Blue Dye Demo protocol has clear (PBS) in well 1 and blue dye in well 2. {% hint style="info" %} [The Opentrons Deck Calibration Video](https://youtu.be/bKYmFyJACaU) has another good demonstration of jogging the pipette tip in the XYZ directions into the center of the engraved "+" symbol on the Opentrons deck is very similar to the [labware calibration for the Omni-Stainer™](https://vimeo.com/779408843) which is performed using the embossed "+" symbol on the C12 or S12 Omni-Stainer™ lid. {% endhint %} ### Troubleshooting 1. Ensure that the blue dye filling and washing is complete 2. Very small pockets of carryover buffer towards the bottom corners of the staining chamber are normal and should not affect staining quality because usually there’s no tissue present there. However, if you wish for a more complete exchange, you may wish to increase the wash volume as necessary. 3. If the flow seems abnormally slow, and the exchange seems incomplete and uneven: 1. Carefully check for bubbles in the chamber. Bubbles may form if the buffers used have significant amounts of dissolved gas. If bubbles are present, slide the sample up and down to release the bubble. If that doesn’t help, re-mount the sample. When in doubt, degas buffers before the run. 4. Make sure that the sample is positioned correctly within the staining chamber. 1. S12 Omni-Stainer: ensure the gray aluminum shelf is correctly positioned in the staining chamber, if this is off the liquid may be applied to the outside of the cover tile rather than into the capillary gap. 2. C12 Omni-Stainer: make sure that the coverslip rests evenly on both spacers, as in this diagram:\ ![](/files/mODBAyQW6e5VrsgiAhQs) ### Omni-Stainer Assembly and Sample Prep reference: {% content-ref url="/pages/NmuN25IFo3T1ISCeUP2R" %} [S12 Assembly and Sample Prep](/product-guides/s12-assembly-and-sample-prep) {% endcontent-ref %} {% content-ref url="/pages/1H3JOB80iwMXtLYKepXk" %} [C12 Assembly and Sample Prep](/product-guides/c12-assembly-and-sample-prep) {% endcontent-ref %} # Software Requirements ### To use Parhelia Omni-Stainer™ you will need: * #### Microsoft Excel [The labware layout .xlsx files can also be viewed and edited in Google Sheets if running the Chrome Browser ](https://support.google.com/docs/answer/9331167?hl=en#2.3) * **Opentrons App** The Opentrons App is available for free download from this a link on this page: ### Parhelia StainWorks™ [Parhelia StainWorks™](https://stainworks.parheliabio.com/) is a web-based application that provides a UI for no-code customization of the protocols and does not require any software installation. Both .xlsx files and .py files are downloaded from StainWorks. Additionally, we currently require users to occasionally download new custom labware layout files (.JSON format) from our GitHub repository at:
### For R\&D: You will want a program where you can read and write Python files, what we recommend\* to our users is: * #### PyCharm The free Community Addition ("CE") can be downloaded by scrolling down to the button of the download page: {% hint style="info" %} You could also use a built-in program like TextEdit for Mac, but it is much harder to actually use it since there is no color coding. Or you could use the official Python viewer IDLE, by downloading Python at . There are web-based applications for this, but I don't know enough about them to recommend any. {% endhint %}
### Recommended, but not required, software for advanced support: * #### TeamViewer For faster support, you may want to get set up with TeamViewer (available for free download at:[ https://www.teamviewer.com/en-us/download/windows/](https://www.teamviewer.com/en-us/download/windows/)) so our team can control the robot directly through your computer, but that is not required. * #### ManyCam If you want to use a webcam then you will want a recording/streaming program. There is a variety of software for this, and what we recommend to our users is ManyCam (available for free download at: )
If anything remains unclear please us know! Email:
# PhenoCycler® (CODEX) PhenoCycler Fusion (PCF) and IHC were the first applications of Parhelia's capillary gap staining automation, so there is a great background of knowledge and support for this particular application. {% hint style="warning" %} :construction: This page is (still) under construction :construction: {% endhint %} ## Intro CODEX (CO-Detection by indEXing), now called [PhenoCyler™](https://www.akoyabio.com/phenocycler/), enables spatial phenotyping of millions of cells, at single cell resolution using cyclic detection of DNA-indexed antibody panels simultaneously targeting up to 100 Protein and RNA Biomarkers. While the imaging process has [achieved great speeds](https://youtu.be/KpljJd0YXwk), the sample prep remains a complicated staining process, consuming many hours. {% hint style="success" %} **Now, with the Omni-Stainer™ system, the entire staining procedure is fully automated, allowing researchers to focus on the science, not the sample prep.** {% endhint %}
## Protocol Setup {% hint style="info" %} If you have not done so already, we recommend users start by completing the Blue Dye Demo Automated Protocol (link below), it covers general information on running a protocol like importing the custom labware definitions and doing tip calibration. {% endhint %} {% content-ref url="/pages/C6Pql6XPM8NNOELpQPXx" %} ["Blue Dye Demo" Automated Protocol](/product-guides/blue-dye-demo-automated-protocol) {% endcontent-ref %} ## Labware Setup As shown in the video above, Labware Layout files contain the necessary information about what labware, instruments and reagents go where, and in what quantities. Our default protocols have been developed to match the steps and reagents of the manual protocols published by Akoya. {% hint style="info" %} The reagents themselves are commercially available through [Akoya Bioscience's assay kits](https://www.akoyabio.com/phenocycler/assays/) {% endhint %} To run PhenoCycler Fusion sample prep on 1-12 samples you will need: * **Reagent Plate**\ \ This is a 96-well plate or 8-well PCR strips held in a rack filled with small volume CODEX reagents. Each column or PCR strip is designated for a single sample. Special care is required for some small volume reagents, like antibody mixes and Reagent F, which should be added on the day of the experiment to avoid evaporation.\ \ A single run can support up to 12 different Antibody Cocktails for 12 different samples. Reagent preparation instructions are included in the Labware Layout file. {% hint style="info" %} In addition to baking, dewax, antigen retrieval, and PCF prep; we also have an optional **"Antibody Screening Mode"** that allows users to apply a single tuple of reporters (without using a PCF flow cell or the PCF instrument) to aid in antibody panel evaluation and background signal mapping. Each sample has it's own dedicated column in the plate so multiple sets of reporters can be tested on replicate samples simultainioussly. {% endhint %} {% hint style="warning" %} One must use fresh Reagent F aliquots every time! In our experience, antibody staining quality can be compromised by using Reagent F aliquots that are nearing expiration. {% endhint %} * **Buffers Reservoir**\ A deep well, sealed, 12-channel reagent trough is used, filled with bulk buffers for all samples. Depending on the experiment's mode and sample type, specific buffers are utilized, detailed in the Labware Layout file. * One fully fillled bulk buffer reservoir (19 ml / per well) is enough to stain up to 12 samples. If the buffers are to be reused – the resrvoir has to be re-sealed after the run. Some bulk reagents inside the 12-trough contain formalidehyde and mixtures of DMSO with water based buffers. We recommend not to store the reservoir for longer than a week – due to stability of these reagents. * **Opentrons Tips (300 μL or 200 μL)**\ Two tip racks are recommended for the protocol * **Parhelia Omni-Stainer™**\ The protocol works with C12 or S12 staining modules in the Opentrons OT-2, with or without the thermal sheath. * **Parhelia Temperature Module**\ While we do offere a version of PCF prep that includes a manual pause step to run PCF staining without a temperature mpdule - the quality and reproducibility of staining results are greatly improved utilizing a temperature module. ## Tissue Preparation ### Fresh-Frozen (FF) tissues 1. Have ready either slides or coverslips pre-treated with poly-L-lysine. It it important to have high quality freshly coated glass media. Best results are observed with the following protocol. Soak slides or coverslips for at least overnight at RT in poly-L-lysine solution (e.g. Sigma-Aldrich P8920-100ML). On a day of making the sections wash the necessary number of slides/coverslips with deionized water (to remove the excess of poly-L-lysine) and dry on filter paper. Dry slides/coverslips can be stored without deterioration of coating for up to several weeks. 2. Make \~7μm-thick fresh-frozen (FF) tissue section and place it onto a coverslip (C12) or glass slide (S12) that has been treated with poly-lysine (video link). This should be done according to conventional histological methods or according to instructions in the video. Note: Once mounted, these fresh-frozen tissue sections can be safely stored for up to several months at −80 °C 3. Remove mounted FF tissue sample from -80. Set the sample on a layer of Drierite dessicant (Thermo Fisher Scientific, cat. no. 07-578-3A) at room temperature until dry (\~2 minutes for s coverslip). 4. Immerse the sample in 10 ml of room temperature acetone for 10 minutes. Slide or coverslips Coplin jars could be used to this end. 5. Allow the specimen to air-dry for 2 minutes at room temperature. 6. Rehydrate and make the flow cell in PBS or S1. Rehydration following the drying step is achieved while coverslip or slide reside in PBS or S1 while the flow cell is being constructed. 1. **For Slide-Mounted Samples** - see step 2 in [**S12 Assembly and Sample Prep**](/product-guides/s12-assembly-and-sample-prep) section. 2. **For Coverslip-Mounted Samples -** see step 2 in [**C12 Assembly and Sample Prep**](/product-guides/c12-assembly-and-sample-prep) section. ### FFPE If a Parhelia Temperature Module is used, baking and dewax happens on the Parhelia Omni-Stainer using our Dewax HIER protocol developed with Akoya. In the automated protocol, the flow cell is constructed dry by simply placing a Cover Pad ontop the slide and loading it in the S12 staining module. For users without a temperature module these are the manual steps prior to running a PCF staining run: 1. Mount a 4-μm-thick FFPE tissue section onto a glass slide. (the sections can be stored practically endlessly at 4C inside a desiccated container) 2. Bake the slide at 70 °C for 1 h. 3. Follow with standard rehydration protocol. E.g. : Incubate the specimen (in a glass Coplin jar) in xylene for 30 min at RT, exchanging the solvent twice. 4. Sequentially place the specimen in a series of solutions, each time for 3 min, at RT in the following order: xylene, xylene, 100% ethanol, 100% ethanol, 95% ethanol, 95% ethanol, 80% ethanol, 70% ethanol, ddH2O and ddH2O (vol/vol). An automated linear stainer can be used for this step. If performing manually, use enough solution to have the coverlsips or slides fully submersed for each step. 5. Assemble the flow cell {% hint style="info" %} Rehydration step is acheived by constructing the Parhelia Omni-Stainer™ flow cell. {% endhint %} ## After PCF staining From this point if the multicycle rendering process is to be done inside the [Phenocycler Fusion ](https://www.akoyabio.com/phenocycler-fusion-system/)– the slide should be processed according to the steps described for mounting of the stained slide for the Phenocycler Fusion protocol. If the multicycle rendering process is to be done inside the original CODEX instrument, the coverslip has to be placed section up into the microscope stage of the original CODEX instrument and further imaged according to the instructions. [CODEX User Manual PDF (see Chapter 8: Use of the CODEX® Instrument)](https://www.akoyabio.com/wp-content/uploads/2021/01/CODEX-User-Manual.pdf) ## Troubleshooting ### Protocol analysis failure: Unable to find a labware definition If a protocol is loaded into the Opentrons App, but the required labware definition is missing the protocol will show up looking like:
And the error details will look like:

Missing labware definition error details

If this happens, import the missing labware definitions, and then click "Reanalyze protocol" in the Protocols tab. For help adding the missing labware definitions check out this page: {% content-ref url="/pages/nKQMxb6PDGLrLArrVpt2" %} [Importing labware definitions](/product-guides/automation-opentrons-ot-2-setup/importing-labware-definitions) {% endcontent-ref %}
# Gram Stain This page is under construction # IF This page is under construction # IHC This page is under construction # H\&E This page is under construction # RNAScope This page is under construction # Opal This page is under construction # IMC This page is under construction # Background info and Lens training docs This section of the Gitbook is primarily for training the Lens AI search tool, and is not really crafted for human absorption. Please follow the Product guides rather than this section. {% hint style="info" %} This is for automation bundles only, click here for manual operation:[Manual Operation and "Blue Dye Demo" Manual Protocol](/product-guides/manual-operation-and-blue-dye-demo-manual-protocol) {% endhint %} {% hint style="info" %} The Parhelia Omni-Stainer™ has a standard SBS footprint and is thus compatible with nearly every robotic liquid handler. However, we develop staining protocols for the Opentrons OT-2 robot because of its affordability, accuracy, and programmability. {% endhint %} #### If you want to get a head start, download our GitHub archive from [here](https://github.com/parheliabio/ot2-scripts/archive/refs/heads/main.zip)! ## Opentrons OT-2 Robot and Software setup {% embed url="" %} ^ Click here ^ For Opentrons Set up {% endembed %} The Opentrons Help Center at is a fantastic resource that has s[tep-by-step guides](https://support.opentrons.com/s/article/Get-started-Unbox-the-OT-2) as well as [**video guides**](https://support.opentrons.com/s/article/Get-started-Unbox-the-OT-2) **(which we strongly recommend) like this video series below** that takes you from box to protocol. {% embed url="" %} ^ This playlist covers ^ 1. [Unboxing the OT-2](https://youtu.be/nvjNHod-2hU) 2. [Unlocking the OT-2](https://youtu.be/c2fnzlFfCSE) 3. [Setting up the Opentrons App](https://youtu.be/sqkvy8xdPVw) 4. [Attaching Pipettes to the OT-2](https://youtu.be/xmCItodWx2o) 5. [Deck Calibration on the OT-2](https://youtu.be/bKYmFyJACaU) 6. [Tip Length & Pipette Offset Calibration on the OT-2 ](https://youtu.be/7mMtvdcV-dM) 7. [Running a Protocol on the OT-2](https://youtu.be/hiN0EOW3vHM) {% hint style="info" %} The Deck Calibration process in video 5 of jogging the pipette tip in the XYZ directions into the center of the engraved "+" symbol on the Opentrons deck is very similar to the labware calibration for the Omni-Stainer™ which is performed using the embossed "+" symbol on the C12 or S12 Omni-Stainer™ lid.\ [As in this video ](https://vimeo.com/779408843) {% endhint %} ## Checklist * [ ] Set up Opentrons OT-2 robot. [This set-up guide is a great reference](https://support.opentrons.com/s/ot2-get-started), and our friends at Opentrons are happy to assist you with this if you have any [questions](https://support.opentrons.com/s/information-about-opentrons-support). * [ ] [Download Opentrons App](https://opentrons.com/ot-app/) * [ ] [Download Parhelia Bio's base OT-2 scripts and labware definitions from GitHub by clicking here](https://github.com/parheliabio/ot2-scripts/archive/refs/heads/main.zip) * [ ] If you would prefer to visit the GitHub and download individual files our GitHub is at * [ ] Download customized or stock Omni-stainer script from [Stainworks](https://3qcwn6iy48.execute-api.us-east-1.amazonaws.com/) * [ ] Load protocols and labware definitions into Opentrons software * [ ] Follow protocol-specific instructions, for preparing and loading your labware and doing protocol setup. We strongly recommend starting with the Blue Dye Demo protocol to get comfortable using the device before conducting actual experiments.

Example of staining protocol loaded into Opentrons

## Step 1 - Place the Omni-Stainer, reagent reservoirs, pipette tip rack, and antibody plate into the dedicated slots on the deck of the liquid handler. When inserting the labware (plates, reservoirs, Omni-Stainers) into the slot of the liquid handler deck it is important to ensure the complete in-flush alignment of the labware bottom with the surface of the deck. Lack of such alignment may result in tips bending while hitting the bottom of the labware wells. SBS labware should click in or rest fully in a deck location, a common error is one of the back corners not being fully inserted and level with the rest of the labware. ## Step 2 - Create a running protocol using the Staining Works Software The Python files with staining protocols for an individual experiment can be easily configured using the Staining Works Software. For auditing and accountability we recommend storing the individual day – specific protocols and assigning them a name consisting of the operator name, date, and the purpose of the experiment. ## Step 2 - Start the experiment. Calibration step. ## Step 3 - Start the experiment. Initiate the protocol run. # Importing labware definitions Custom labware definitions have been meticulously created for Parhelia Omni-stainer instruments and reservoirs, these files are essential for running Parhelia Bio protocols. Labware definitions are special files in JSON format that contain the information about the geometry of labwares (number of wells, XY positioning, well depth etc) that the Opentron OT-2 robot needs to correctly access the samples. We created custom labware definitions that describe Parhelia Omni-stainer instruments and reservoirs (deep-well 12-trough, 96-well black plate and 96-well skirted plate) that we use in our OT-2 protocols. Download our GitHub archive from [here](https://github.com/parheliabio/ot2-scripts/archive/refs/heads/main.zip), unzip, and import the JSON files from the labware folder into the Opentrons OT-2 as detailed[ in this guide](https://support.opentrons.com/s/article/Using-labware-in-your-protocols). Labware definitions are specialized JSON format files that contain essential information about the labware's geometry, such as the number of wells, XY positioning, and well depth. These files are crucial for enabling the Opentron OT-2 robot to accurately access the samples. To facilitate this process, we have meticulously crafted custom labware definitions specifically for Parhelia Omni-stainer instruments and reservoirs. These definitions encompass various labware types, including deep-well 12-trough, 96-well black plate, and 96-well skirted plate, which are extensively employed in our OT-2 protocols. To get started, simply download our GitHub archive from the [here](https://github.com/parheliabio/ot2-scripts/archive/refs/heads/main.zip), unzip the files, and follow the comprehensive guide to import the JSON files from the labware folder into the Opentrons OT-2 as detailed[ in this guide](https://support.opentrons.com/s/article/Using-labware-in-your-protocols). Click below if you wish to download just the labware definition files: {% file src="/files/RQHiRYAFhFdZ7YSJeyP8" %} After downloading, unzip the archive and follow this guide for how to import the custom labware definitions: Labware definitions are specialized JSON format files containing crucial information about labware geometry. These files are essential for accurate sample handling by the Opentron OT-2 robot. Custom labware definitions have been meticulously created for Parhelia Omni-stainer instruments and reservoirs. Various labware types, including deep-well 12-trough, 96-well black plate, and 96-well skirted plate, are extensively used in OT-2 protocols. To get started, download the GitHub archive, unzip the files, and follow the comprehensive guide to import the JSON files into your Opentrons OT-2 system. # StainWorks software: a centralized app for accessing and configuring protocols ## Step 1 - Creating a high humidity environment inside the Omni-Stainer Place at least 30 ml of deionized water into the Omni-Stainer. This helps maintaining humid environment and preventing evaporation from the covertile/slide or pad/coverlsip sandwich ![](https://images.unsplash.com/photo-1522881451255-f59ad836fdfb?crop=entropy\&cs=tinysrgb\&fm=jpg\&ixid=MnwxOTcwMjR8MHwxfHNlYXJjaHw0fHx3cml0ZXxlbnwwfHx8fDE2NjA1ODc5Nzk\&ixlib=rb-1.2.1\&q=80) ## Step 2 - Using a coverslip with a sample to construct the flowcell Both the S(slide) and the C(overslip) Omni-Stainers utilize so called "capillary liquid exchange " concept which is based on creating a thin layer of staining solution evenly covering the sample. The space where the sample is facing the exchangeable reactive staining solutions is called a "flow cell". To create the flow cell in C type Omni-Stainer the coverslip with the sample facing down needs to placed onto an omni-pad. This can be achieved in a number of ways – the easiest one by doing it under the layer of PBS or any such compatible buffer inside a tip box lid or a beaker (see the video below). As confidence and experience are built this can also be done by placing a drop of compatible buffer on top of the omni-pad and then covering the drop with a coverslip such that bubbles are avoided. In the case bubbles are encountered – sliding the coverslip such that bubble is released and then sliding it back after adding a bit more of the liquid allows to "expel" the bubbles ![](https://images.unsplash.com/photo-1515378791036-0648a3ef77b2?crop=entropy\&cs=tinysrgb\&fm=jpg\&ixid=MnwxOTcwMjR8MHwxfHNlYXJjaHw2fHxwb3N0fGVufDB8fHx8MTY2MDU4ODAzMg\&ixlib=rb-1.2.1\&q=80) # Reagent Layout files Labware layout.xlsx file # Running the "Blue Dye Demo" protocol # Copy of CODEX (PhenoCyler™) Omni-Stainer + OT-2 automates the entire 5.5 hr (fresh-frozen tissue sections) or 7 hr (FFPE) CODEX (PhenoCyler™) staining process with ease and precision. With optional antibody screening mode! {% hint style="warning" %} :construction: This page is under construction :construction: {% endhint %} **Akoya Biosciences' PhenoCycler™ system**, formerly known as CODEX (CO-Detection by indEXing), is a revolutionary spatial biology technology that provides single-cell resolution for highly multiplexed analysis of 100+ biomarkers. This is achieved through the cyclic detection of DNA-indexed antibody panels. In the complex world of multicellular organisms, no cell exists in isolation. For instance, in the tumor microenvironment, 50% of cells are not tumor cells. Traditional histology attempts to image these intricate interactions one marker at a time, while genetic screening provides deep multidimensionality to resolve cell populations but loses the spatial context. PhenoCycler™ bridges this gap, offering both spatial context and the multidimensionality of omics approaches. **Parhelia Biosciences' Omni-Stainer™ system** automates the sample preparation process of the PhenoCycler™ system, making it more efficient and reliable. The PhenoCycler™-Fusion, touted as "The Fastest Single-Cell Spatial Biology Solution," enables users to "map a million cells in 10 minutes." However, the sample preparation between tissue sections and the PhenoCycler fluidics devices remains a delicate staining process that takes at least 7 hours for FFPE samples or 5.5 hours for fresh frozen samples. {% hint style="warning" %} Previously, this staining protocol was a manual task, requiring meticulous care to avoid damaging valuable samples on extremely fragile glass coverslips. This complex procedure followed an extensive 100+ page manual, consuming many hours. {% endhint %} {% hint style="success" %} **Now, with the Omni-Stainer™ system, the entire staining procedure is fully automated, allowing researchers to focus on the science, not the sample prep.** {% endhint %}

How the Omni-Stainer fits into the PhenoCycler (CODEX) Workflow

No more tiptoeing around fragile glass coverslips or thumbing through a 100+ page manual. {% embed url="" %} How to generate and run a custom CODEX staining protocol on an Opentrons OT-2 + Parhelia Bio Omni-Stainer {% endembed %} ## Akoya Fluidics Devices {% embed url="" %} PhenoCycler-Fusion Product Video {% endembed %} {% embed url="" %} PhenoCycler fluidics instrument {% endembed %} ## Parhelia Biosciences {% embed url="" %} Omni-Stainer Product Video {% endembed %} ## CODEX protocol (work in progress) CODEX protocol
Reagents: 1. S1/\[Hydration buffer + 1.6% PFA] (PBS with 0.5%BSA, EDTA) (Goltsev et al 2018, Cell) 2. Preblock: Staining Buffer (S2) with SS DNA and rabbit and mouse IgGs. Preblock is the solution that is used to dilate the antibodies into and to stain the section. 3. Plating buffer: R1 with ssDNA and HOechst 4. H2/CODEX Buffer
Protocol:
1. Slide or coverslip with FFPE or fresh frozen section. 1. Frozen section pretreatment 1. Pull out of -80 freezer dry/bring to RT on Drye Rite 2. Drop the section into RT acetone for 10 min 3. Dry the sample on a filter paper 2. Rehydrate (PBS, S1). Note – should not contain any detergents. When the protocol is done on the robot – the rehydration step is substituted by the mounting procedure (preparing flow cell). The slide or coverslip are dipped into the reservoir with hydration buffer (PBS/Hydration buffer) and are either positioned over the Support Pad or covered with a Cover Pad \######################################### Starting this step the protocol is done robotically. \######################################### 4. Postfix (S1 with 1.6%PFA; PBS with 4% PFA) for 10min. Note – should not contain any detergents. This step is skipped if the FFPE sample is to be stained. 5. Wash/Equilibrate in Staining Buffer (S2) 30min. 6. Preblock (solutions from the row A of the 96 well plate are pipetted, called CODEX® Blocking Buffer in CODEX user Manual) for 15min at RT 7. Dispense the Stain (solutions from the second row of the 96 well plate are pipetted). 8. Optionally the protocol is paused here to allow the user to place the OMNI-stainer into the cold room or refrigerator 9. Unless the #8 is used – incubate for 3h at RT for fresh frozen or for 8 hours at RT for FFPE. 10. Wash 3 times with S2 11. Fix in Storage (High Salt) buffer with 4% PFA. 12. Wash in PBS 13. Apply RT methanol and incubate for 10 min 14. Wash with PBS 15. Postfixation with BS3: Dilute the F-reagent in PBS and pipet onto the section( PBS from the 12-trough is pipetted into the third row of the 96 well plate and this mix is dispensed into the section) 16. Wash with PBS 17. If the screening mode is False the protocol stops here with placing the section into the storage buffer. \
From this point if the multicycle rendering process is to be done inside the Phenocycler Fusion – the slide should be processed according to the steps described for mounting of the stained slide for the Phenocycler FUsion protocol.
If the multicycle rendering process is to be done inside the original CODEX robot the coverslip has to be placed section up into the microscope stage of the original CODEX robot and further imaged according to the instructions. \
18. If the screening mode is True 19. The section is “stripped” (iterative washes with R1(screening 20%DMSO in H2) \[Screening buffer (16mL 1x CODEX buffer mixed with 4mL DMSO)]and R2(stripping 80%DMSO in H2) buffers) 20. The section is rendered: detector oligos (in plating buffer) from the fourth row of the 96 well plate are pipetted onto the section and incubated for 10 min 21. The section is washed in R1 22. The section is stored in the Storage buffer.
\######################################### That is the end of the robotic protocol \#########################################
To image the slide or coverslip – wash in CODEX buffer, optionally pre-stain with DAPI/Hoechst for 1 min (these steps could be done manually in the OMNI-stainer or on in the 6-well plate) and either mount in CODEX buffer with nail polish or in any permanent Fluoromount system followed by imaging ([section 6.3 of CODEX User Manual](https://www.akoyabio.com/wp-content/uploads/2021/01/CODEX-User-Manual.pdf))
# CODEX Reagent Setup The following is an excerpt from Black et al., 2021, Nature Protocols Black, S., Phillips, D., Hickey, J. W., Venkataraaman, V. G., Samusik, N., Goltsev, Y., Schürch, C. M., & Nolan, G. P. (2021). CODEX multiplexed tissue imaging with DNA-conjugated antibodies. Nature Protocols, 16(8), 3802-3835. ## Reagent setup PBS-Tween solution Prepare a 0.1% (vol/vol) Tween solution in 1× DPBS. Store at room temperature (RT: 18–26 °C) for ≤6 months. 500 mM TCEP stock solution Dissolve 717 mg of TCEP in 2.5 ml of ddH2O, adjust to pH 7.0 with sodium hydroxide and bring to a volume of 5 ml with ddH2O. Store at 4 °C for ≤1 year. 2.5 mM TCEP solution for antibody reduction Add 5 μl of 500 mM TCEP and 5 μl of 500 mM EDTA (pH 8.0) to 990 μl of 1× DPBS. Mix by gently pipetting up and down. Prepare fresh before each experiment. 5 M NaCl solution Dissolve 146.1 g of NaCl in 500 ml of ddH2O. Add 0.02 % (wt/vol) NaN3 to this solution. Store at RT for ≤1 year. Buffer C Add 1 ml of 1 M Tris (pH 7.0), 1 ml of 1 M Tris (pH 7.5), 30 ml of 5 M NaCl solution and 2 ml of 500 mM EDTA (pH 8.0) to 966 ml of ddH2O. Add 200 mg of NaN3 for a 0.02% (wt/vol) concentration in this solution. Store at RT for ≤1 year. High-salt buffer C Add 20 μl of 5 M NaCl solution to 380 μl of buffer C. Mix gently by pipetting up and down. Prepare fresh before each experiment. High-salt PBS Add45mlof5MNaClsolutionand25mlof10×DPBSto180mlofddH2O.Add50mgofNaN3 for a 0.02% (wt/vol) concentration in this solution. Store at RT for ≤1 year. Stock antibody stabilizer solution Add 0.02% (wt/vol) NaN3 to Candor PBS antibody stabilizer solution. Store at 4 °C for ≤1 year. CODEX antibody stabilizer solution Add 1 ml of 5 M NaCl solution and 100 μl of 500 mM EDTA (pH 8.0) to 9 ml of stock antibody stabilizer solution. Vortex briefly to mix. Store at 4 °C for ≤1 year. Antigen retrieval solution, pH 9 Dilute Dako target retrieval solution at a 1:10 ratio with ddH2O (vol/vol). Prepare fresh before each experiment. 70% ethanol solution Dilute 100% ethanol in ddH2O to make a 70% (vol/vol) solution. Store at RT for ≤3 months. 80% Ethanol Dilute 100% ethanol in ddH2O to make a 80% (vol/vol) solution. Store at RT for ≤3 months. 95% Ethanol Dilute 100% ethanol in ddH2O to make a 95% (vol/vol) solution. Store at RT for ≤3 months. 1× PBS for antigen retrieval Add 150 μl of 10× PBS to 1.35 ml of ddH2O. Store at RT for ≤6 months. TBS IHC wash buffer with Tween 20 Dilute 20× TBS IHC wash buffer with Tween 20 at a 1:20 ratio with ddH2O (vol/vol). Store at RT for ≤1 month. CODEX staining buffer 1 (S1) Add5mlof500mMEDTA(pH8.0),50mlof10×DPBS,2.5gofBSAand100mgofNaN3 to 445 ml of ddH2O. Gently mix. Store at 4 °C for ≤1 year. 1 M Na2HPO4 Dissolve 70.98 g of Na2HPO4 in 500 ml of ddH2O. Add 0.02% (wt/vol) NaN3 to this solution. Store at RT for ≤1 year. 1 M NaH2PO4 Dissolve 69 g of NaH2PO4•H2O in 500 ml of ddH2O. Add 0.02% (wt/vol) NaN3 to this solution. Store at RT for ≤1 year. CODEX staining buffer 2 (S2) Add250mlofS1,30.5mlof1MNa2HPO4,19.5mlof1MNaH2PO4 and25mlof5MNaCl solution to 175 ml of ddH2O. Adjust the pH to 6.8–7.0 with sodium hydroxide. Gently mix. Store at 4 °C for ≤1 year. Blocking reagent 1 (B1) Dissolve 10 mg of mouse IgG in 10 ml of S2. Store in 0.5-ml aliquots at 4 °C for ≤1 year. Blocking reagent 2 (B2) Dissolve 10 mg of rat IgG in 10 ml of S2. Store in 0.5-ml aliquots at 4 °C for ≤1 year. Blocking reagent 3 (B3) sheared salmon sperm DNA This reagent comes at a 10-mg/ml concentration in ddH2O at −20 °C. Store in 0.5-ml aliquots at 4 °C for ≤1 year. 1× Tris-EDTA (TE) buffer Add 1 ml of 1 M Tris (pH 8.0), 200 μl of 500 mM EDTA (pH 8.0) and 0.02% (wt/vol) NaN3 to 98.8 ml of ddH2O. Store at RT for ≤1 year. BC4 solution Prepare a mixture of the 57 nonmodified CODEX DNA oligonucleotides (Supplementary Table 1) by dissolving in TE buffer to a final concentration of 0.5 mM per oligonucleotide. Store in 0.5-ml aliquots at 4 °C for ≤1 year. CODEX FFPE blocking solution Add 50 μl of B1, 50 μl of B2, 50 μl of B3 and 70 μl of BC4 to 780 μl of S2. Mix gently by pipetting up and down. Store at 4 °C for ≤2 weeks. CODEX staining buffer 4 (S4) Add 50 ml of 5 M NaCl solution to 450 ml of S1. Gently mix. Store at 4 °C for ≤1 year. Paraformaldehyde fixation solution\ Dilute 16% (wt/vol) paraformaldehyde (PFA) at a 1:10 ratio with S4 (vol/vol). Prepare fresh before each experiment and use a fresh vial of 16% PFA every 2 weeks. BS3 aliquots Dissolve 50 mg of BS3 in 250 μl of DMSO (ampule). Store in 20-μl aliquots at −20 °C for ≤6 months. Final fixative solution\ Add 20 μl of BS3 (thawed to RT) to 1 ml of 1× DPBS. Mix gently by pipetting up and down. Prepare fresh before each experiment. H2 buffer Add 30 ml of 5 M NaCl solution, 10 ml of 1 M Tris (pH 7.5), 0.943 ml of Triton X-100, 2.03 g of MgCl2•6H2O and 0.02% (wt/vol) NaN3 to 960 ml of ddH2O. Store at RT for ≤1 year. Hybridization buffer Combine 100 ml of DMSO with 400 ml of H2 buffer. Stir gently to thoroughly combine. Prepare fresh before each experiment. Stripping buffer Add 62.5 ml of H2 buffer to 187.5 ml of DMSO. Stir gently to thoroughly combine. Prepare fresh before each experiment. CODEX hybridization solution For each tissue section, add 7 μl of B3, 1 μl of each complementary fluorescent oligonucleotide and the appropriate volume of H2 buffer to a final volume of 100 μl. Mix gently in an Eppendorf tube by pipetting up and down. Prepare fresh before each experiment. Hoechst staining solution Add 1 μl of Hoechst 33342 to 999 μl of H2 buffer. Mix gently in an Eppendorf tube by pipetting up and down. Prepare fresh before each experiment. Plate buffer Add 83.3 μl of Hoechst 3342 and 2.5 ml of B3 to 50 ml of H2 buffer. Store in the dark at 4 °C for ≤4 weeks. Fluorescent oligonucleotide stock solution Dissolve the lyophilized fluorescent oligonucleotide pellet in the appropriate volume of 1× TE buffer to a concentration of 100 μM (note: volume provided by IDT). Store in the dark at −20 °C for ≤2 years. Generally, we will order custom fluorescent reporter oligonucleotides from IDT that are HPLC purified, 1-μmol batch size and lyophilized. Fluorescent oligonucleotide working solution Dilute the fluorescent oligonucleotide stock solution at a 1:10 ratio with 1× TE buffer (vol/vol). Store in the dark at 4 °C for ≤1 year. DRAQ5 staining solution Add 2.5 μl of DRAQ5 to 247.5 μl of plate buffer. Prepare fresh when setting up the multicycle plate. # Temperature-controlled incubations ## Step 1 - Creating a high humidity environment inside the Omni-Stainer Place at least 30 ml of deionized water into the Omni-Stainer. This helps maintaining humid environment and preventing evaporation from the covertile/slide or pad/coverlsip sandwich ## Step 2 - Using a coverslip with a sample to construct the flowcell Both the S(slide) and the C(overslip) Omni-Stainers utilize so called "capillary liquid exchange " concept which is based on creating a thin layer of staining solution evenly covering the sample. The space where the sample is facing the exchangeable reactive staining solutions is called a "flow cell". To create the flow cell in C type Omni-Stainer the coverslip with the sample facing down needs to placed onto an omni-pad. This can be achieved in a number of ways – the easiest one by doing it under the layer of PBS or any such compatible buffer inside a tip box lid or a beaker (see the video below). As confidence and experience are built this can also be done by placing a drop of compatible buffer on top of the omni-pad and then covering the drop with a coverslip such that bubbles are avoided. In the case bubbles are encountered – sliding the coverslip such that bubble is released and then sliding it back after adding a bit more of the liquid allows to "expel" the bubbles # Page 2 # ParheliaBio.com content Training Data and content from https\://parheliabio.com/ # Advanced Tissue Staining Solutions | ParheliaBio #### Say goodbye to variability, reagent waste, and months of painstaking training. #### Welcome spatial sample prep automation at a push of a button ### Parhelia Omni-Stainer™: ### Modular platform for spatial assay automation ### Our customers ### Capillary Gap Staining ### and Laminar Flow Buffer Exchange Capillary ### Air Displacement Pipetting * Zero Dead Volume * Onboard Mixing * Disposable Tips * Microliter Precision (0.6 μL) Pipette Untitled video - Made with Clipchamp ### Heating and Cooling The Omni-Stainer™ is made of heat-conductive aluminum and can be used in conjunction with a temperature control module, allowing the temperature to be programmatically controlled between 4C and 95C. This enables a variety of protocols, such as FFPE Antigen Retrieval, smRNA-FISH, or CycIF to be performed directly inside the Omni-Stainer™ device. #### Our Automated Protocols ### Parhelia Bio supports biomedical research and innovation **Researchers creating innovative products for researchers** Parhelia icon data **sample data** Peace of mind of with reproducible results. Parhelia icon clinical solutions **clinical solutions** We accommodate diverse laboratory and research needs, including slide or coverslip staining. Parhelia icon knowledge **knowledge & resources** We help small or underfunded research and diagnostic facilities. Parhelia icon team **meet your expert team** We are passionate about supporting academic research. **EFFICIENT - ACCURATE - AFFORDABLE** #### Book a meeting today Virtual Demo Complete your details to book a meeting and view our virtual demonstration of the Omni-Stainer™ **What people are saying...** With the Parhelia Omni-Stainer I’ve been able to increase the number of coverslips or slides I can do CODEX staining at the same time and, on top of that, I’ve been able to save reagents and antibody while preserving staining quality. Using their product was a great investment for our project #### Ruan Medrano, Ph.D **Washington University in St. Louis School of Medicine** We were very excited to be an early-access adopter of Parhelia’s automated coverslip staining solution. Their product significantly improved the reproducibility, labor, and throughput of our smFISH and CasFISH workflows. The Parhelia team provided easy to implement software scripts to run the protocols on the OT-2, and were very responsive in answering any of our questions as we were first setting up the process. Finally, the low upfront capital acquisition cost versus other cell staining automation solutions makes it very easy to justify the ROI. #### Un Kwon-Casado **Dahlia Biosciences - Co-Founder and CEO** I am not so sure about this Parhelia thing… I’d rather buy a machine from a big vendor that takes up the whole room, guzzles gallons of proprietory antibodies per run and costs 50X more. #### Said Nobody **Ever** Testimonial 1 # Omni-Stainer™ S12 - Automation Bundle * [Description](broken://pages/N3tC5eXTZ4Qu0hYIXsBD) * [Essential Addons: Heating Samples? Yes](broken://pages/N3tC5eXTZ4Qu0hYIXsBD) * [Essential Addons: Heating Samples? No](broken://pages/N3tC5eXTZ4Qu0hYIXsBD) * [Technical Support & Resources](broken://pages/N3tC5eXTZ4Qu0hYIXsBD) ### Description The Omni-Stainer![™](https://ci4.googleusercontent.com/proxy/OA_iKlYBZ3tMmgyfzB29d4g7HycHDFk8kSRoBLDaoj5NvfBkTrbsRHATEF6pFvsycGgweh88DrZr=s0-d-e1-ft#https://emojis.superhuman.com/2122.png) S12, Automation Bundle includes an S12 Omni-Stainer, 4 support shelves, lid for automation, 10 Parhelia Biosciences’ proprietary Coverpads, 3-pack of Humidity Barrier Paper, predeveloped protocols and a 5 ml bottle of Parhelia Biosciences’ Conditioning Reagent. The S12 is to be used with Parhelia Biosciences’ proprietary Coverpads sold in a 48 unit pack and standard glass slides (not included in the bundle). **Essential Addons:** **Will you be heating samples?**\ If **Yes,** you will need [Coverpads – 48 Pack](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FAXeF6XyzahXjaZY8jNXa%2Fcover%20pad%2048%20pack?alt=media) and [S12 Thermal Sheath](broken://pages/ynWd3BwSKZ0i9oVIjU78) If **No**, you will need [Coverpads – 48 Pack](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FAXeF6XyzahXjaZY8jNXa%2Fcover%20pad%2048%20pack?alt=media) ### Essential Addons: Heating Samples? Yes **Will you be heating samples?**\ If **Yes,** you will need [Coverpads – 48 Pack](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FAXeF6XyzahXjaZY8jNXa%2Fcover%20pad%2048%20pack?alt=media) and [S12 Thermal Sheath](broken://pages/ynWd3BwSKZ0i9oVIjU78) ### Essential Addons: Heating Samples? No If **No**, you will require [Coverpads – 48 Pack](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FAXeF6XyzahXjaZY8jNXa%2Fcover%20pad%2048%20pack?alt=media) Do you require a Liquid Handler? If so, please contact us for guidance on the [Opentrons products](broken://pages/nTBIBpLVHQGjPv8tdZdA) # Omni-Stainer™ S12 - Manual Operation Bundle ### Description The Omni-Stainer![™](https://ci4.googleusercontent.com/proxy/OA_iKlYBZ3tMmgyfzB29d4g7HycHDFk8kSRoBLDaoj5NvfBkTrbsRHATEF6pFvsycGgweh88DrZr=s0-d-e1-ft#https://emojis.superhuman.com/2122.png) S12, Manual Operation Bundle includes an S12 Omni-Stainer, 4 support shelves, lid for manual use, 10 Parhelia Biosciences’ proprietary Coverpads, predeveloped protocols and a 5 ml bottle of Parhelia Biosciences’ conditioning reagent. The S12 is to be used with Parhelia Biosciences’ proprietary Coverpads sold in a 48 unit pack and standard glass slides (not included in the bundle). **Essential Addons:**\ You will need Coverpads to use this product.\ Please order – [Coverpads – 48 Pack](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2F5pfuIqpIW3QintKtBNGR%2Fcover%20Pad%2048%20pack?alt=media) ### Essential Addons You will need Coverpads to use this product.\ Please order – [Coverpads – 48 Pack](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FAXeF6XyzahXjaZY8jNXa%2Fcover%20pad%2048%20pack?alt=media) # Shop ### Compact, Affordable Flexible and Easy To Use We are passionate about supporting academic research by providing cost-effective, precision, customisable tissue staining automation. ### Product Categories * [Product Bundles (6)](broken://pages/WrPp2OFk2mHEroK9wvpc) * [Consumables (5)](broken://pages/ZdOs5FXbG7Tk3an2yOvl) * [Opentrons Robots (11)](broken://pages/nTBIBpLVHQGjPv8tdZdA) * [Software (2)](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2F5tuMGVlWuGy695xgGfgc%2Fsoftware?alt=media) ### Product Bundles * [Quick View](broken://pages/N3tC5eXTZ4Qu0hYIXsBD) ### [Omni-Stainer™ S12 – Automation Bundle](broken://pages/N3tC5eXTZ4Qu0hYIXsBD) $4,995.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](broken://pages/iFde5HQSAxnrnPwRtdSd) ### [Omni-Stainer™ S12 – Manual Operation Bundle](broken://pages/iFde5HQSAxnrnPwRtdSd) $4,200.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](broken://pages/IYVd1chrppcE14gWTpvh) ### [Omni-Stainer™ S12 Thermal Sheath](broken://pages/IYVd1chrppcE14gWTpvh) $1,750.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](broken://pages/uA8W6OonHpdrRaJTJSBS) ### [Omni-Stainer™ C12 – Automation Bundle](broken://pages/uA8W6OonHpdrRaJTJSBS) $4,800.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](broken://pages/nWOUQHAKxDNu77r0hbUq) ### [Omni-Stainer™ C12 – Manual Operation Bundle](broken://pages/nWOUQHAKxDNu77r0hbUq) $4,000.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](broken://pages/ynWd3BwSKZ0i9oVIjU78) ### [Omni-Stainer™ C12 Thermal Sheath](broken://pages/ynWd3BwSKZ0i9oVIjU78) $1,750.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * ### Consumables * [Quick View](broken://pages/Cd8kv2swcQn292BWsJS2) ### [Conditioning Reagent 5ml Bottle](broken://pages/Cd8kv2swcQn292BWsJS2) $64.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FAXeF6XyzahXjaZY8jNXa%2Fcover%20pad%2048%20pack?alt=media) ### [S12 Coverpads – 48 Pack](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FAXeF6XyzahXjaZY8jNXa%2Fcover%20pad%2048%20pack?alt=media) $525.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FNnUTeHknxWH7n4Lp3XXV%2Fsupport%20pad%2048%20pack?alt=media) ### [C12 Support Pad – 48 Pack](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FNnUTeHknxWH7n4Lp3XXV%2Fsupport%20pad%2048%20pack?alt=media) $525.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](broken://pages/GxoWkuYGMvPYtZtVZEO2) ### [S12 Filter Paper](broken://pages/GxoWkuYGMvPYtZtVZEO2) $33.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](broken://pages/o8tqak8IjOLNtgCl5Rbr) ### [C12 Filter Paper](broken://pages/o8tqak8IjOLNtgCl5Rbr) $33.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * ### Software * [Quick View](https://parheliabio.com/product/custom-protocol-development/) ### [Custom Protocol Development](https://parheliabio.com/product/custom-protocol-development/) $999.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](https://parheliabio.com/product/stainworks-software/) ### [StainWorks™](https://parheliabio.com/product/stainworks-software/) $650.00 / Year and 90 Days free trial [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * ### Opentrons Robots * [Quick View](broken://pages/6Oe3F0fR8ZET5VO4dWBo) ### [Opentrons OT-2 Liquid Handling Robot](broken://pages/6Oe3F0fR8ZET5VO4dWBo) $6,500.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](broken://pages/vDBbCSJRcMoFAW2bTpqR) ### [Opentrons Single-Channel Electronic Pipette p20 (GEN2)](broken://pages/vDBbCSJRcMoFAW2bTpqR) $1,250.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](broken://pages/Y7nYCCR9ZvmVKXtzmiBx) ### [Opentrons 300 µl Pipette Tip Tip Racks – 9600 tips](broken://pages/Y7nYCCR9ZvmVKXtzmiBx) $550.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](https://parheliabio.com/product/opentrons-multi-channel-electronic-pipette-p300/) ### [Opentrons Multi-Channel Electronic Pipette p300 (GEN2)](https://parheliabio.com/product/opentrons-multi-channel-electronic-pipette-p300/) $2,000.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](https://parheliabio.com/product/opentrons-temperature-module/) ### [Opentrons Temperature Module](https://parheliabio.com/product/opentrons-temperature-module/) $3,750.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](https://parheliabio.com/product/opentrons-single-channel-electronic-pipette-p300/) ### [Opentrons Single-Channel Electronic Pipette p300 (GEN2)](https://parheliabio.com/product/opentrons-single-channel-electronic-pipette-p300/) $1,250.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](https://parheliabio.com/product/opentrons-single-channel-electronic-pipette-p1000/) ### [Opentrons Single-Channel Electronic Pipette p1000 (GEN2)](https://parheliabio.com/product/opentrons-single-channel-electronic-pipette-p1000/) $1,250.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](https://parheliabio.com/product/opentrons-20-%c2%b5l-pipette-tip-rack-and-refill/) ### [Opentrons 20 µl Pipette Tip Racks – 9600 tips](https://parheliabio.com/product/opentrons-20-%c2%b5l-pipette-tip-rack-and-refill/) $550.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](https://parheliabio.com/product/opentrons-1000-%c2%b5l-pipette-tip-racks-9600-tips/) ### [Opentrons 1000 µl Pipette Tip Racks – 9600 tips](https://parheliabio.com/product/opentrons-1000-%c2%b5l-pipette-tip-racks-9600-tips/) $550.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * [Quick View](https://parheliabio.com/product/opentrons-multi-channel-electronic-pipette-p20-gen2/) ### [Opentrons Multi-Channel Electronic Pipette p20 (GEN2)](https://parheliabio.com/product/opentrons-multi-channel-electronic-pipette-p20-gen2/) $2,000.00 [Add to cart](broken://pages/wak6hWIFIEHXdvKNfiHm) * **EFFICIENT - ACCURATE - AFFORDABLE** #### View our virtual demo Virtual Demo Complete your details and view our virtual demonstration of the Omni-Stainer™ Name(Required) Email(Required) Organisation(Required) Name This field is for validation purposes and should be left unchanged. # Omni-Stainer™ S12 Thermal Sheath [Home](broken://pages/DHike9v7J81CxGxzWi62) / [Product Bundles](broken://pages/WrPp2OFk2mHEroK9wvpc) / Omni-Stainer™ S12 Thermal Sheath $1,750.00 S12 Thermal Sheath insulates the Omni-Stainer![™](https://ci4.googleusercontent.com/proxy/OA_iKlYBZ3tMmgyfzB29d4g7HycHDFk8kSRoBLDaoj5NvfBkTrbsRHATEF6pFvsycGgweh88DrZr=s0-d-e1-ft#https://emojis.superhuman.com/2122.png) during heating of samples Omni-Stainer™ S12 Thermal Sheath quantity [ADD TO QUOTE](broken://pages/IYVd1chrppcE14gWTpvh) SKU: 40003 Category: [Product Bundles](broken://pages/WrPp2OFk2mHEroK9wvpc) * [Description](broken://pages/IYVd1chrppcE14gWTpvh) * [Technical Support & Resources](broken://pages/IYVd1chrppcE14gWTpvh) ### Description The S12 thermal sheath insulates the Omni-Stainer![™](https://ci4.googleusercontent.com/proxy/OA_iKlYBZ3tMmgyfzB29d4g7HycHDFk8kSRoBLDaoj5NvfBkTrbsRHATEF6pFvsycGgweh88DrZr=s0-d-e1-ft#https://emojis.superhuman.com/2122.png) S12 while heating samples. The product comes with a shutter that can be used in conjunction with a liquid handling robot to open and close as needed to execute a protocol. It can also be used while manually processing samples. Please visit our Support & Knowledge Base here >> ### Related products * [Omni-Stainer™ C12 – Manual Operation Bundle**0** out of 5$4,000.00 ](broken://pages/nWOUQHAKxDNu77r0hbUq)[Add to cart](broken://pages/IYVd1chrppcE14gWTpvh) * [Omni-Stainer™ C12 Thermal Sheath**0** out of 5$1,750.00 ](broken://pages/ynWd3BwSKZ0i9oVIjU78)[Add to cart](broken://pages/IYVd1chrppcE14gWTpvh) * [Omni-Stainer™ C12 – Automation Bundle**0** out of 5$4,800.00 ](broken://pages/uA8W6OonHpdrRaJTJSBS)[Add to cart](broken://pages/IYVd1chrppcE14gWTpvh) * [Omni-Stainer™ S12 – Automation Bundle**0** out of 5$4,995.00 ](broken://pages/N3tC5eXTZ4Qu0hYIXsBD)[Add to cart](broken://pages/IYVd1chrppcE14gWTpvh) # Omni-Stainer™ C12 - Automation Bundle * [Description](broken://pages/uA8W6OonHpdrRaJTJSBS) * [Essential Addons: Heating Samples? Yes](broken://pages/uA8W6OonHpdrRaJTJSBS) * [Essential Addons: Heating Samples? No](broken://pages/uA8W6OonHpdrRaJTJSBS) * [Technical Support & Resources](broken://pages/uA8W6OonHpdrRaJTJSBS) ### Description The Omni-Stainer![™](https://ci4.googleusercontent.com/proxy/OA_iKlYBZ3tMmgyfzB29d4g7HycHDFk8kSRoBLDaoj5NvfBkTrbsRHATEF6pFvsycGgweh88DrZr=s0-d-e1-ft#https://emojis.superhuman.com/2122.png) C12, Automation Bundle includes a C12 Omni-Stainer, lid for automation,10 Parhelia Biosciences’ proprietary Support Pads, a 3-pack of Humidity Barrier Paper, predeveloped protocols and a 5 ml bottle of conditioning reagent. The C12 is to be used with Parhelia Biosciences’ proprietary Support Pads and standard coverslips – 48 Pack (Not included in this bundle). **Essential Addons:** **Will you be heating samples?**\ If **Yes,** you will need [Support Pad- 48 Pack](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FNnUTeHknxWH7n4Lp3XXV%2Fsupport%20pad%2048%20pack?alt=media) and [C12 Thermal Sheath](broken://pages/ynWd3BwSKZ0i9oVIjU78) If **No**, you will need [Support Pad – 48 Pack](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FNnUTeHknxWH7n4Lp3XXV%2Fsupport%20pad%2048%20pack?alt=media) ### Essential Addons: Heating Samples? Yes **Will you be heating samples?**\ If **Yes,** you will need [Support Pad – 48 Pack](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FNnUTeHknxWH7n4Lp3XXV%2Fsupport%20pad%2048%20pack?alt=media) and [C12 Thermal Sheath](broken://pages/ynWd3BwSKZ0i9oVIjU78) ### Essential Addons: Heating Samples? No If **No**, you will require [Support Pads – 48 Pack](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FNnUTeHknxWH7n4Lp3XXV%2Fsupport%20pad%2048%20pack?alt=media) Do you require a **Liquid Handler**? If so, please contact us for guidance on the [Opentrons products](https://parhelia.wpengine.com/product-category/opentrons-robot-purchase/) # Omni-Stainer™ C12 Thermal Sheath [Home](broken://pages/DHike9v7J81CxGxzWi62) / [Product Bundles](broken://pages/WrPp2OFk2mHEroK9wvpc) / Omni-Stainer™ C12 Thermal Sheath $1,750.00 C12 Thermal Sheath insulates the Omni-Stainer![™](https://ci4.googleusercontent.com/proxy/OA_iKlYBZ3tMmgyfzB29d4g7HycHDFk8kSRoBLDaoj5NvfBkTrbsRHATEF6pFvsycGgweh88DrZr=s0-d-e1-ft#https://emojis.superhuman.com/2122.png) during heating of samples Omni-Stainer™ C12 Thermal Sheath quantity [ADD TO QUOTE](broken://pages/ynWd3BwSKZ0i9oVIjU78) SKU: 40102 Category: [Product Bundles](broken://pages/WrPp2OFk2mHEroK9wvpc) * [Description](broken://pages/ynWd3BwSKZ0i9oVIjU78) * [Technical Support & Resources](broken://pages/ynWd3BwSKZ0i9oVIjU78) ### Description The proprietary C12 thermal sheath insulates the Omni-Stainer![™](https://ci4.googleusercontent.com/proxy/OA_iKlYBZ3tMmgyfzB29d4g7HycHDFk8kSRoBLDaoj5NvfBkTrbsRHATEF6pFvsycGgweh88DrZr=s0-d-e1-ft#https://emojis.superhuman.com/2122.png) C12 while heating samples. The product includes a shutter that can be used in conjunction with a liquid handling robot to open and close as needed to execute a protocol. It can also be used while manually processing samples. Please visit our Support & Knowledge Base here >> ### Related products * [Omni-Stainer™ C12 – Manual Operation Bundle**0** out of 5$4,000.00 ](broken://pages/nWOUQHAKxDNu77r0hbUq)[Add to cart](broken://pages/ynWd3BwSKZ0i9oVIjU78) * [Omni-Stainer™ C12 – Automation Bundle**0** out of 5$4,800.00 ](broken://pages/uA8W6OonHpdrRaJTJSBS)[Add to cart](broken://pages/ynWd3BwSKZ0i9oVIjU78) * [Omni-Stainer™ S12 – Automation Bundle**0** out of 5$4,995.00 ](broken://pages/N3tC5eXTZ4Qu0hYIXsBD)[Add to cart](broken://pages/ynWd3BwSKZ0i9oVIjU78) * [Omni-Stainer™ S12 – Manual Operation Bundle**0** out of 5$4,200.00 ](broken://pages/iFde5HQSAxnrnPwRtdSd)[Add to cart](broken://pages/ynWd3BwSKZ0i9oVIjU78) # Consumables Archives ### Compact, AffordableFlexible and Easy To Use
We are passionate about supporting academic research by providing cost-effective, precision, low throughput, customisable tissue staining automation. # Conditioning Reagent 5ml Bottle ### Precision tissue staining [Home](broken://pages/DHike9v7J81CxGxzWi62) / [Consumables](broken://pages/ZdOs5FXbG7Tk3an2yOvl) / Conditioning Reagent 5ml Bottle $64.00 5 ml bottle of Parhelia Biosciences’ Conditioning Reagent Conditioning Reagent 5ml Bottle quantity [ADD TO QUOTE](broken://pages/Cd8kv2swcQn292BWsJS2) SKU: 20000 Category: [Consumables](broken://pages/ZdOs5FXbG7Tk3an2yOvl) * [Description](broken://pages/Cd8kv2swcQn292BWsJS2) * [Technical Support & Resources](broken://pages/Cd8kv2swcQn292BWsJS2) ### Description Conditioning Reagent to be used while processing samples in the Omni-Stainer C12 and S12 Please visit our Support & Knowledge Base here >> ### Related products * [C12 Support Pad – 48 Pack**0** out of 5$525.00 ](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FNnUTeHknxWH7n4Lp3XXV%2Fsupport%20pad%2048%20pack?alt=media)[Add to cart](broken://pages/Cd8kv2swcQn292BWsJS2) * [C12 Filter Paper**0** out of 5$33.00 ](broken://pages/o8tqak8IjOLNtgCl5Rbr)[Add to cart](broken://pages/Cd8kv2swcQn292BWsJS2) * [S12 Filter Paper**0** out of 5$33.00 ](broken://pages/GxoWkuYGMvPYtZtVZEO2)[Add to cart](broken://pages/Cd8kv2swcQn292BWsJS2) * [S12 Coverpads – 48 Pack**0** out of 5$525.00 ](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FAXeF6XyzahXjaZY8jNXa%2Fcover%20pad%2048%20pack?alt=media)[Add to cart](broken://pages/Cd8kv2swcQn292BWsJS2) # Omni-Stainer™ C12 - Manual Operation Bundle ### Description The Omni-Stainer![™](https://ci4.googleusercontent.com/proxy/OA_iKlYBZ3tMmgyfzB29d4g7HycHDFk8kSRoBLDaoj5NvfBkTrbsRHATEF6pFvsycGgweh88DrZr=s0-d-e1-ft#https://emojis.superhuman.com/2122.png) C12, Manual Operation Bundle includes a C12 Omni-Stainer, lid for manual use, 10 Parhelia Biosciences’ proprietary Support Pads, predeveloped protocols and a 5 ml bottle of conditioning reagent. The C12 is to be used with Parhelia Biosciences’ proprietary Support Pads and standard coverslips – Pack of 48 (Not included in this bundle). **Essential Addons:**\ You will need Support Pads to use this product.\ Please order – [Support Pads – Pack of 48](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FNnUTeHknxWH7n4Lp3XXV%2Fsupport%20pad%2048%20pack?alt=media) ### Essential Addons **Essential Addons:**\ You will need Support Pads to use this product.\ Please order – [Support Tiles – Pack of 48](https://parhelia.wpengine.com/product/support-pad-48-pack/) # S12 Coverpads - 48 Pack ### Precision tissue staining 48 disposable Coverpads required for use of the Omni-Stainer S12 * [Description](broken://pages/9qEtOyMVA8YWf3z71s7H) * [Technical Support & Resources](broken://pages/9qEtOyMVA8YWf3z71s7H) ### Description 48 Omni-Stainer proprietary disposable Coverpads required for use with the Omni-Stainer S12, the Coverpads will be used in combination with a standard microscopy slide (microscopy slides not included). # C12 Filter Paper ### Precision tissue staining [Home](broken://pages/DHike9v7J81CxGxzWi62) / [Consumables](broken://pages/ZdOs5FXbG7Tk3an2yOvl) / C12 Filter Paper $33.00 C12 Filter Paper, 3 sheet pack C12 Filter Paper quantity [ADD TO QUOTE](broken://pages/o8tqak8IjOLNtgCl5Rbr) SKU: 40401 Category: [Consumables](broken://pages/ZdOs5FXbG7Tk3an2yOvl) * [Description](broken://pages/o8tqak8IjOLNtgCl5Rbr) * [Technical Support & Resources](broken://pages/o8tqak8IjOLNtgCl5Rbr) ### Description 3 pack of filter paper for the Omni-Stainer C12, to be used with the automation lid. Please visit our Support & Knowledge Base here >> ### Related products * [Conditioning Reagent 5ml Bottle**0** out of 5$64.00 ](broken://pages/Cd8kv2swcQn292BWsJS2)[Add to cart](broken://pages/o8tqak8IjOLNtgCl5Rbr) * [S12 Filter Paper**0** out of 5$33.00 ](broken://pages/GxoWkuYGMvPYtZtVZEO2)[Add to cart](broken://pages/o8tqak8IjOLNtgCl5Rbr) * [S12 Coverpads – 48 Pack**0** out of 5$525.00 ](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FAXeF6XyzahXjaZY8jNXa%2Fcover%20pad%2048%20pack?alt=media)[Add to cart](broken://pages/o8tqak8IjOLNtgCl5Rbr) * [C12 Support Pad – 48 Pack**0** out of 5$525.00 ](https://3271682270-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FWKyUXOqIKtqE7cAUE2yQ%2Fuploads%2FNnUTeHknxWH7n4Lp3XXV%2Fsupport%20pad%2048%20pack?alt=media)[Add to cart](broken://pages/o8tqak8IjOLNtgCl5Rbr) # Opentrons Robots Archives ### Compact, AffordableFlexible and Easy To Use
We are passionate about supporting academic research by providing cost-effective, precision, low throughput, customisable tissue staining automation. # S12 Filter Paper ### Precision tissue staining * [Description](broken://pages/GxoWkuYGMvPYtZtVZEO2) * [Technical Support & Resources](broken://pages/GxoWkuYGMvPYtZtVZEO2) ### Description 3 pack of filter paper for the Omni-Stainer S12, to be used with the automation lid # C12 Support Pad - 48 Pack ### Precision tissue staining * [Description](broken://pages/MYr7uUHPPrgeyYgYXr6R) * [Technical Support & Resources](broken://pages/MYr7uUHPPrgeyYgYXr6R) ### Description 48 Omni-Stainer proprietary disposable Support Pads required to be used with coverslips (coverslips not included) in the Omni-Stainer C12 # Opentrons OT-2 Liquid Handling Robot ### Precision tissue staining The high-precision OT-2 lab robot is a flexible, open-source, and user-friendly liquid handler. * [Description](broken://pages/6Oe3F0fR8ZET5VO4dWBo) * [Technical Support & Resources](broken://pages/6Oe3F0fR8ZET5VO4dWBo) ### Description The high-precision OT-2 lab robot is a flexible, open-source, and user-friendly liquid handler.\ Configurable 11-deck platform with options for Magnetic, Temperature and Thermocycler Modules\ Swappable single & 8-channel pipettes for multiple volume configurations\ Standardized (SBS) deck slots allow you to use labware you already own\ Accuracy: 15% at 1uL\ Precision: 5% at 1uL\ Plug-and-play setup with access to protocol libraries to enable immediate assay implementation\ 100% customizable and open to modifications adjustable to your assay\ Modify or customize protocols with our simple yet powerful Python API\ Digital protocol commands surpass manual pipetting capabilities **Pipettes are not included** # Opentrons Single-Channel Electronic Pipette p20 (GEN2) ### Precision tissue staining [Home](broken://pages/DHike9v7J81CxGxzWi62) / [Opentrons Robots](broken://pages/nTBIBpLVHQGjPv8tdZdA) / Opentrons Single-Channel Electronic Pipette p20 (GEN2) $1,250.00 Single-Channel Electronic Pipette p20 (GEN2) Opentrons Single-Channel Electronic Pipette p20 (GEN2) quantity [ADD TO QUOTE](broken://pages/vDBbCSJRcMoFAW2bTpqR) SKU: 70005 Category: [Opentrons Robots](broken://pages/nTBIBpLVHQGjPv8tdZdA) * [Description](broken://pages/vDBbCSJRcMoFAW2bTpqR) * [Technical Support & Resources](broken://pages/vDBbCSJRcMoFAW2bTpqR) ### Description Opentrons Pipettes are designed for lab automation and optimized for use with the OT-2:\ Accurate: comparable specs to pipettes 10x as expensive Please visit our Support & Knowledge Base here >> ### Related products * [Opentrons Temperature Module**0** out of 5$3,750.00 ](https://parheliabio.com/product/opentrons-temperature-module/)[Add to cart](broken://pages/vDBbCSJRcMoFAW2bTpqR) * [Opentrons Single-Channel Electronic Pipette p300 (GEN2)**0** out of 5$1,250.00 ](https://parheliabio.com/product/opentrons-single-channel-electronic-pipette-p300/)[Add to cart](broken://pages/vDBbCSJRcMoFAW2bTpqR) * [Opentrons 20 µl Pipette Tip Racks – 9600 tips**0** out of 5$550.00 ](https://parheliabio.com/product/opentrons-20-%c2%b5l-pipette-tip-rack-and-refill/)[Add to cart](broken://pages/vDBbCSJRcMoFAW2bTpqR) * [Opentrons 300 µl Pipette Tip Tip Racks – 9600 tips**0** out of 5$550.00 ](broken://pages/Y7nYCCR9ZvmVKXtzmiBx)[Add to cart](broken://pages/vDBbCSJRcMoFAW2bTpqR) # Opentrons 300 µl Pipette Tip Tip Racks - 9600 tips ### Precision tissue staining Opentrons 300µL Tips. Optimized for volumes 5µL to 300µL Clear Tips, Polypropylene * [Description](broken://pages/Y7nYCCR9ZvmVKXtzmiBx) * [Technical Support & Resources](broken://pages/Y7nYCCR9ZvmVKXtzmiBx) ### Description Opentrons 300µL Tips. Optimized for volumes 5µL to 300µL Clear Tips, Polypropylene. These tips are DNAse/RNAse, pyrogen and protease free – they are sterilized from ebeam irradiation. Not autoclavable. # Greater Tissue Staining Workflow Efficiency. Slide Staining Kits **Precision tissue staining every time**\ **at the push of a button** ### How it works Greater workflow efficiency - saves time and reduces the need for dedicated personnel and training - automated liquid handlers are faster than manual pipetting. **making tissue auto-staining accessible to all** ### Peace of mind of having reproducible results – with simple or complex protocols ### Omni-Stainer™ C12 for coverslips * Compact: footprint of a 96-well plate * Stains up to 12 coverslip-mounted samples at a time * Saves reagents: 50% less reagents compared to open-top staining * Never lose or break a sample * Built-in wet chamber * Use it by hand or insert into any SBS-compatible liquid handler to automate your staining ![Omni-stainer-C12-with-tip-open-lid-3](https://parheliabio.com/wp-content/uploads/bb-plugin/cache/Omni-stainer-C12-with-tip-open-lid-3-square.png) ### How it works for coverslips how-it-works-coverslips 1. Coverslip with sample rests on 50um thin spacers, forming a capillary chamber. Staining reagents are supplied by a liquid handler (or by hand!) 2. Sample is incubated in a thin capillary layer 3. When the sample is washed, the old one is instantly displaced by the **laminar flow** ![Omni-stainer-S12-with-samples](https://parheliabio.com/wp-content/uploads/bb-plugin/cache/Omni-stainer-S12-with-samples-square.jpg) ### Omni-Stainer™ S12 for slides * Compact: footprint of a 96-well plate * Stains up to 12 coverslip-mounted samples at a time * Saves reagents: 50% less reagents compared to open-top staining * Never lose or break a sample * Built-in wet chamber * Use it by hand or insert into any SBS-compatible liquid handler to automate your staining how-it-works-for-slides * The same capillary displacement principle, but the sample is mounted on a slide and covered with a coverpad that incorporates capillary spacers **EFFICIENT - ACCURATE - AFFORDABLE** #### View our virtual demo Virtual Demo Complete your details and view our virtual demonstration of the Omni-Stainer™ ### Placing slides into Omni-Stainer™ **Slide assembly**\ This video shows how to assemble the slide/coverpad sandwich and place it into the slide Omni-StainerTM. First the tacky spacers are exposed by removing the protective tape from the cover pad. To avoid airbubbles between the slide and the coverpad the slide is first placed in the small tray with compatible buffer (water or PBS) and in such submerged stage is covered with a coverpad. The spacers are tacky enough to hold the coverpad in place (after slightly pressed against the slide) but yet when wet allow repositioning the coverpad over the slide. After the sandwich is assembled the combo is placed on the shelf and then into the Omni-StainerTM **Coverslip Assembly**\ This video shows how to assemble the coverslip/support pad sandwich and place it into the coverslip Omni-StainerTM system. To avoid airbubbles between the slide and the support pad, a drop of compatible buffer (water or PBS) is first placed onto the support pad. The coverslip with a sample is then placed face down on the pad to avoid bubble formation and ensure that the liquid fully fills the flow cell formed by sandwiching the support pad and the coverslip. After the sandwich is assembled the combo is placed into the coverslip version of the Omni-StainerTM system. ### Manual staining Using Omni-Stainer™ platform by hand Omni-Stainer™ S12 platform Demo Video Omni-Stainer™ C12 platform Demo Video The Omni-StainerTM platform can be used as a device for manual staining. In such case the solutions are exchanged by manual pipetting. After the pipetting the Omni-StainerTM system should be closed with a lid. In such configuration it can be placed into the 4C refrigerator or into the heated incubator. Before the start of any staining experiment 20-30ml of water should be placed into the Omni-StainerTM system to create the humid chamber environment which helps to reduce the evaporation of staining solutions from the slide/coverpad or coverslip/support pad sandwich.” ### Automated Staining Demo using Opentrons OT-2 Omni-Stainer™ S12 platform automation demo Omni-Stainer™ C12 platform Demo Video For automated liquid exchange the Omni-Stainer™ system has to be place into programmable liquid handler (e.g. Opentrons). The design of the lid including the prewetted perforated filter paper on the lid ensures minimizing the evaporation of staining reagents from the slide/coverpad or coverslip/support pad sandwich. Staining automation can be attained by inserting the Omni-Stainer™ into an SBS-compatible liquid handler, and how we are partnering with Opentrons and adopting their versatile ultra-low-cost liquid robot OT-2 to deliver complete staining automation solutions. As a part of our ‘automation package’, we provide pre-written protocols for various assays, including H\&E, IHC, IF, CycIF, CODEX, FFPE antigen retrieval, CAS-FISH, IMC, RNAscope. Custom protocol development services are available. **Omni-Stainer™ is compatible with most liquid handling robots but if you don't have one, we recommend Opentrons OT-2** Protocols Supported on OT-2: * IF * IHC * CODEX single and multi-strain * Cas-FISH * FFPE Antigen Retrieval (HIER) * Opal **Coming soon:** * smRNA-FISH * Rolling Circle Amplification * Cyclic IF * IMC & MIBI * and many more... OT 2 ### Heating and cooling on the robot: FFPE-IHC **Omni-Stainer™ system rests on top of the OT-2 thermal module after:**\ Human tonsil FFPE antigen retrieval at 95°C\ Anti-CD8 Primary antibody staining at 4°C\ Secondary antibody staining, DAB and hematoxylin at room temperature Heating and cooling on the robot: FFPE-IHC #### Are you ready for the Omni-Stainer™ platform to transform your life? **What people are saying...** With the Parhelia Omni-Stainer I’ve been able to increase the number of coverslips or slides I can do CODEX staining at the same time and, on top of that, I’ve been able to save reagents and antibody while preserving staining quality. Using their product was a great investment for our project #### Ruan Medrano, Ph.D **Washington University in St. Louis School of Medicine** We were very excited to be an early-access adopter of Parhelia’s automated coverslip staining solution. Their product significantly improved the reproducibility, labor, and throughput of our smFISH and CasFISH workflows. The Parhelia team provided easy to implement software scripts to run the protocols on the OT-2, and were very responsive in answering any of our questions as we were first setting up the process. Finally, the low upfront capital acquisition cost versus other cell staining automation solutions makes it very easy to justify the ROI. #### Un Kwon-Casado **Dahlia Biosciences - Co-Founder and CEO** I am not so sure about this Parhelia thing… I’d rather buy a machine from a big vendor that takes up the whole room, guzzles gallons of proprietory antibodies per run and costs 50X more. #### Said Nobody **Ever** Testimonial 1 # Opentrons setup guides Content from support.opentrons.com # Opentronssetupguides ### Disclaimer This Copy is from Opentrons and is Copyrighted, © 2023 Opentrons, it is available at [https://support.opentrons.com/](https://www.google.com/url?q=https://support.opentrons.com/\&sa=D\&source=editors\&ust=1684531643375924\&usg=AOvVaw3jYd5jzWq91C5EHn5_3bQL) , if you have any questions or concerns just email Citation ### Citation: Opentrons. (2023). How Labware Offsets work on the OT-2. Retrieved May 19, 2023, from [https://support.opentrons.com/s/article/How-Labware-Offsets-work-on-the-OT-2](https://www.google.com/url?q=https://support.opentrons.com/s/article/How-Labware-Offsets-work-on-the-OT-2\&sa=D\&source=editors\&ust=1684531643377172\&usg=AOvVaw0R7nrAKslJk7YZCFnrGYZo) ### How Labware Offsets work on the OT-2 ### Learn how Labware Offset data is utilized during the Labware Position Check workflow when running a protocol on the OT-2. In this article: 1. What is a Labware Offset? 2. What is Labware Position Check? 3. Should I always use Labware Position Check? 4. How do I run Labware Position Check? ### What is a Labware Offset? A Labware Offset is data that helps the OT-2 determine what small adjustments need to be made for a pipette to go to the right place on a specific labware in a specific deck slot. Labware Offset data is unique to a specific combination of labware definition, deck slot, and OT-2. To create Labware Offset data, you perform Labware Position Check before running your protocol. As of version 6.0 of the Opentrons App, you can apply previously calculated offsets — even across different protocols — as long as they are for the same type of labware in the same deck slot on the same robot. ### What is Labware Position Check? Labware Position Check is the recommended guided workflow that Labware Offsets are created through (which are used to increase the precision of your protocol). When you run Labware Position Check, the OT-2’s will move to the top center of the A1 well of each piece of labware. If the pipette nozzle or tip is not centered, you can reveal the OT-2’s jog controls to make adjustments. Whatever adjustment you make will be applied to all wells on the labware in that slot. Offset data is an x, y, z vector measured to the nearest 0.1 mm. As of software version 6.0.0, the Labware Offset data created through the Labware Position check is stored on the OT-2. Since it is now local to the OT-2, it is possible to apply previous Labware Offsets — even across different protocols — as long as they are for the same type of labware in the same deck slot on the same robot. ### Should I always use Labware Position Check? Opentrons recommends using Labware Position Check before every protocol run, but it is not mandatory. If you have saved Labware Offset data and apply it before running Labware Position Check, the pipette will start at those saved positions — you only have to double-check their accuracy. Labware Position Check is intended to correct for minor variances. Opentrons does not recommend using Labware Position Check to compensate for large positional adjustments. Needing to set large labware offsets could indicate a problem further upstream, such as bad deck or pipette calibration, or an incorrect labware definition. We recommend [checking your OT-2's calibration health](https://www.google.com/url?q=https://support.opentrons.com/s/article/Check-your-OT-2-s-calibration-health\&sa=D\&source=editors\&ust=1684531643380741\&usg=AOvVaw1qCHouRz8_2_jHW2vei0D-) to first determine which components might need further attention. ### How do I run Labware Position Check? The Opentrons App will recommend that you run Labware Position Check as part of the setup for your protocol. After [selecting the protocol you want to run, then choosing to run it on a specific OT-2](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Run-your-protocol\&sa=D\&source=editors\&ust=1684531643381741\&usg=AOvVaw1PBo0P5K93t9BuhiI1vc6d), you’ll be taken to the protocol setup page. Under Step 2: Labware Setup, click Run Labware Position Check to begin the guided workflow. ![Run Labware Position Check.png](/files/lxZVAzqy25pl6xgXcMrG) Follow the guided workflow in the app to check labware positions. If needed, reveal the jog controls and adjust the position of the pipette.
![Check Labware Opentrons 96 tip rack.png](/files/jhYG9pByAlxvB1N9pKSo) \ As you jog the pipette, the app will show updated Labware Offset values.![Screen Shot 2022-07-28 at 1.47.15 PM.png](/files/NReZBlpAGhAOaIDUERiX)\ When you have aligned the pipette properly on all labware, click Close and Apply Labware Offset Data. At this point, you can proceed to run your protocol.![Screen Shot 2022-07-28 at 1.45.50 PM.png](/files/Ho6PfshWi7HR2EbnbBbI) After this, you can select Proceed to run (with applied Labware Offset data) or go through the Labware Position Check again to reset or change Labware Offsets. ![labwareoffsets04.png](/files/9mEaKE9f5zJ9UJp1adjE) When your run is complete, you can view the Labware Offset data that was applied to the run by clicking the chevron next to the run record on the robot’s device detail page. Labware Offset data for up to 20 runs are stored on the robot, and can be accessed and applied from the same computer or another computer connecting to the same robot. \ ![Screen Shot 2022-07-28 at 2.01.23 PM.png](/files/V9vqsyum2Ixah8pOb2nm)
If you re-run your protocol (or run a protocol that uses the same type of labware, in the same slot, on the same robot), the app will automatically prompt you to apply your previously stored Labware Offset data. ![apply-labware-offsets](/files/MeInsTMy8oZWVTmg4w5u) You can either use those exact offsets, or if you run Labware Position Check again they will be used as the starting point for jogging the pipette, rather than 0.00 on each axis. Live chat: Chat with an Expert Copyright © 2023 Opentrons ⌘J Skip to Main Content Opentrons Help Center * [Home](https://www.google.com/url?q=https://support.opentrons.com/s/\&sa=D\&source=editors\&ust=1684531643385419\&usg=AOvVaw0hJY9NMEH6xQR60eIViMom) * Topics Expand search * [OT-2: CALIBRATION](https://www.google.com/url?q=https://support.opentrons.com/s/topic/0TO2S000002ZMjrWAG/ot2-calibration\&sa=D\&source=editors\&ust=1684531643386039\&usg=AOvVaw30CfUncWzhT_1hyRR25eMt) ### Troubleshooting calibration issues ### Troubleshooting calibration issues Important Note: the information in this article regarding the tip probe applies only to users running software released before version 4.0.0 There are two types of issues related to calibration: technical issues that block the calibration processes and technical issues related to the robot's movement during a protocol run. ### Issues during calibration process #### Pipette calibration failure The most common issues during pipette calibration are: * Pipette colliding with the tip probe * Pipette missing the tip probe If the pipette is missing the tip probe: 1. Complete a[ ](https://www.google.com/url?q=https://support.opentrons.com/s/article/Factory-resetting-the-OT-2\&sa=D\&source=editors\&ust=1684531643387581\&usg=AOvVaw0OzLhbxbkz1ECXziA5QXV1)[factory reset](https://www.google.com/url?q=https://support.opentrons.com/s/article/Factory-resetting-the-OT-2\&sa=D\&source=editors\&ust=1684531643387919\&usg=AOvVaw1tl1EqhlfNSXj14SIbDOMc) of pipette offset calibration and retry[ ](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Calibrate-tip-length-and-pipette-offset\&sa=D\&source=editors\&ust=1684531643388248\&usg=AOvVaw353754I2ouYpvJTeKVALaS)[pipette offset calibration](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Calibrate-tip-length-and-pipette-offset\&sa=D\&source=editors\&ust=1684531643388614\&usg=AOvVaw2PNOcDLnMIBkSiAARI-ddU) 2. If the issue persists, complete another factory reset of pipette offset calibration and deck calibration and[ ](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Calibrate-the-deck\&sa=D\&source=editors\&ust=1684531643389105\&usg=AOvVaw0TUrqHgJ0NoZsuoeCFJrKv)[redo deck calibration](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Calibrate-the-deck\&sa=D\&source=editors\&ust=1684531643389373\&usg=AOvVaw1hSpFPkyX7k7B59CxJWiXI) (make sure you use Opentrons tips for deck calibration). Then retry pipette offset calibration. 3. If the issue persists, contact technical support, let the team know the troubleshooting steps you've tried so far and a video of the failed calibration, and request the next steps. If the pipette has collided with the tip probe: 1. First visually check all the switches on the tip probe to confirm the switches are in the proper orientation and that they haven't sustained any damage (observe reference images below to determine proper orientation). 2. Complete steps 1-3 above. Improper orientation: ![tip\_probe\_bad\_.jpg](/files/Cpqmwo2qxzIC6WI7tbBp) Proper orientation: ![tip\_probe\_good\_.jpg](/files/yEp3pHkGIBfsnwxRILfi) #### Labware Position Check Failure The most common issue to occur during the labware position check is a crash. This is a severe issue, as crashing causes[ corrupt calibration data](https://www.google.com/url?q=https://support.opentrons.com/s/article/How-Labware-Offsets-work-on-the-OT-2\&sa=D\&source=editors\&ust=1684531643390969\&usg=AOvVaw0wpEhtgjsf6afiubZO5lUJ). If you experience a crash during labware position check, reupload your protocol and restart the labware position check. You should investigate for the following root causes: * Does the labware definition loaded in the protocol match the physical labware in the slot? * Did you complete pipette offset calibration? If you have completed the above and are still having issues, follow these steps in order: 1. Complete a[ ](https://www.google.com/url?q=https://support.opentrons.com/s/article/Factory-resetting-the-OT-2\&sa=D\&source=editors\&ust=1684531643391702\&usg=AOvVaw2FL1hNHH9TClNumc6K5XGa)[factory reset](https://www.google.com/url?q=https://support.opentrons.com/s/article/Factory-resetting-the-OT-2\&sa=D\&source=editors\&ust=1684531643392023\&usg=AOvVaw3_JYrj-_ugcetQmdTJ7zmh) of pipette offset calibration 2. Complete[ ](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Calibrate-the-deck\&sa=D\&source=editors\&ust=1684531643392396\&usg=AOvVaw1ugD-n3hEEuVOKem__y7HX)[deck calibration](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Calibrate-the-deck\&sa=D\&source=editors\&ust=1684531643392653\&usg=AOvVaw03O4WFMNQTyuFCXwr3ZpAE) (make sure to use Opentrons tips) 3. Reupload your protocol and retry calibration If there is a specific labware that the pipette is colliding against during the labware position check, we recommend the following workaround: 1. Before commanding the pipette to move to that particular labware, remove that labware from the deck 2. Command the pipette to move to the empty slot 3. Jog the pipette so there is room to put the labware back in its slot 4. Jog the pipette to position and calibrate as normal ### Calibration issues during a run #### Troubleshooting Movement Issues There are many reasons your OT-2 can move erratically during a run. It is important to understand that troubleshooting movement issues like this can require a good deal of upfront investigation to diagnose and resolve. Ultimately, if you are experiencing issues and are troubleshooting (whether independently or with assistance from technical support), it's a best practice to capture images/video of the behavior and clarify the following details: * What commands in the protocol are being executed at the time of the crash? * Which pipette is experiencing the crash? * Which mount is that pipette on? * Which labware is the pipette crashing against? * How is this labware loaded in the protocol? * What is the expected behavior? Having images/videos of the behavior as well as a strong command of the above details related to your issue will help significantly in the troubleshooting process, especially if you reach out to technical support. #### Crashing During a Run Robot crashing refers to when the robot crashes pipettes into labware or crashes the entire pipette carriage against its own physical boundaries during a run. The first most common reason for crashing during a run is corrupt calibration data. If you did not complete labware position check or pipette calibration before the run, we suggest you reupload the protocol and complete these calibration processes. Furthermore, ensure that 1) the labware is properly mounted in the slot, and 2) during the calibration process, that there are no collisions, neither between the pipettes and the labware, nor the pipette carriage with its physical boundaries. Rerun the protocol and observe whether the issue persists. If the issue does persist, you may follow these steps for a more exhaustive method of rewriting calibration data: 1. Complete a[ ](https://www.google.com/url?q=https://support.opentrons.com/s/article/Factory-resetting-the-OT-2\&sa=D\&source=editors\&ust=1684531643395037\&usg=AOvVaw2b9onfST0JTkP0j59B90eP)[factory reset](https://www.google.com/url?q=https://support.opentrons.com/s/article/Factory-resetting-the-OT-2\&sa=D\&source=editors\&ust=1684531643395387\&usg=AOvVaw1Vazu514R0VOJnb16Cl0k5) of pipette offset calibration 2. Complete a[ ](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Calibrate-the-deck\&sa=D\&source=editors\&ust=1684531643395886\&usg=AOvVaw0fYYir4AR--X-DPJCs3FhX)[deck calibration](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Calibrate-the-deck\&sa=D\&source=editors\&ust=1684531643396204\&usg=AOvVaw09mJGmPTzkhRECP5evNf2U) 3. Reupload your protocol and complete[ ](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Calibrate-tip-length-and-pipette-offset\&sa=D\&source=editors\&ust=1684531643396634\&usg=AOvVaw3fBb450zOqU2jGMQlaCNBl)[pipette offset calibration](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Calibrate-tip-length-and-pipette-offset\&sa=D\&source=editors\&ust=1684531643396926\&usg=AOvVaw1BzstFBeREZTLnl4twqKD-) and[ ](https://www.google.com/url?q=https://support.opentrons.com/s/article/How-Labware-Offsets-work-on-the-OT-2\&sa=D\&source=editors\&ust=1684531643397203\&usg=AOvVaw3uFRSSyy8lesj_FBs4D3xI)[labware position check](https://www.google.com/url?q=https://support.opentrons.com/s/article/How-Labware-Offsets-work-on-the-OT-2\&sa=D\&source=editors\&ust=1684531643397442\&usg=AOvVaw37fucL91a1bmXe7Gn7x9UC) 4. Observe whether the erratic behavior persists The second most common reason for crashing is that the defined labware in the protocol does not match the physical labware on the deck. To verify, you should check your protocol to identify the definition being used, and confirm through our[ ](https://www.google.com/url?q=https://labware.opentrons.com/\&sa=D\&source=editors\&ust=1684531643398051\&usg=AOvVaw2rrtgB4lqNIBZRD7FcFQe9)[Labware Library](https://www.google.com/url?q=https://labware.opentrons.com/\&sa=D\&source=editors\&ust=1684531643398335\&usg=AOvVaw0uXkVni35MvaUVGKIDEkQL) that the definition matches the labware on the deck. For instructions on how to use the library, refer to the following article:[ ](https://www.google.com/url?q=https://support.opentrons.com/s/article/Using-the-Labware-Library\&sa=D\&source=editors\&ust=1684531643398680\&usg=AOvVaw1R3CLNuXr0hwAN1MyS5HKQ)[Using the Labware Library](https://www.google.com/url?q=https://support.opentrons.com/s/article/Using-the-Labware-Library\&sa=D\&source=editors\&ust=1684531643399018\&usg=AOvVaw2G8qhnkvquDG_TIJ-WMxjo). Finally, the root cause of the issue may not be that the robot is crashing in error, but that an error in the protocol is commanding the robot to crash against labware or against its physical boundaries. In this case, or if the above troubleshooting did not resolve your issue, contact the support team and we will work to 1) identify whether the crashing issue is reproducible on a robot in our office, and 2) work with you to resolve your issues. #### Positional Accuracy Issues with positional accuracy refer to the pipette's failure to move to the calibrated position in a well during a run. This is a relatively uncommon movement issue, and you should seek assistance from technical support to troubleshoot and resolve these types of issues. #### Pipette Drifting Pipette drifting refers to when a pipette moves further from the center of wells as it iterates through a column/row of a piece of labware. The two most common root causes for this behavior are mismatched labware definitions and corrupt deck calibration. To investigate the mismatched labware definition issue, you should investigate whether the labware definition loaded in your protocol matches the physical labware on the deck. To troubleshoot deck calibration, you should redo deck calibration, reupload the protocol, redo pipette and labware calibration, and rerun the protocol. If the issue persists, contact technical support at . This Copy is from Opentrons and is Copyrighted, © 2023 Opentrons, it is available at [https://support.opentrons.com/](https://www.google.com/url?q=https://support.opentrons.com/\&sa=D\&source=editors\&ust=1684531643400673\&usg=AOvVaw3312WqYhGU4vNqEN9FABjB) , if you have any questions or concerns just email ⌘J # GetstartedImportaprotocol ### Citation OpenTrons. (2023). Get started: Import a protocol. OpenTrons Support. Retrieved from [https://support.opentrons.com/s/article/Get-started-Import-a-protocol](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol\&sa=D\&source=editors\&ust=1684532198667225\&usg=AOvVaw1MBR3Bb8MIAs5kCAEl5KHr) URL: [https://support.opentrons.com/s/article/Get-started-Import-a-protocol](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol\&sa=D\&source=editors\&ust=1684532198668104\&usg=AOvVaw2xgiWC2lq6wX0qp_vjYYkg) * ### Title: Get started: Import a protocol After you’ve attached pipettes and finished deck, tip length, and pipette offset calibrations, you can import a protocol. [Introduction](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol%23Intro\&sa=D\&source=editors\&ust=1684532198669548\&usg=AOvVaw1JluM2jTjpQ2mc2S-4ihnI) [Before starting](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol%23Before_starting\&sa=D\&source=editors\&ust=1684532198670039\&usg=AOvVaw1aNWWOuAIyEP0X5cVG8_Cu) [Procedure](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol%23Procedure\&sa=D\&source=editors\&ust=1684532198670476\&usg=AOvVaw1BANwRh9fOO88LB-Mdi6QV) [Step 1: Import Python (.py) or Protocol Designer (.json) file](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol%23Step_1\&sa=D\&source=editors\&ust=1684532198670949\&usg=AOvVaw3qGMBjk0IFgjHHN8h7pdXx) [Troubleshooting: “Module not found” or “File not found” errors during protocol analysis](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol%23TS_Protocol_analysis\&sa=D\&source=editors\&ust=1684532198671491\&usg=AOvVaw10UyScRfv7ZyDW7ZVgB2MH) [Step 2: Select protocol](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol%23Step_2\&sa=D\&source=editors\&ust=1684532198671994\&usg=AOvVaw2BQPvq3Nod-BG4HgDHAV4i) [Step 3: Proceed to run setup](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol%23Step_3\&sa=D\&source=editors\&ust=1684532198672508\&usg=AOvVaw0lNACoQruN7ojCBKn_JYWG) [Troubleshooting: Help! I don't see any available robots listed / I'm unable to connect](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol%23TS_Unable_to_connect\&sa=D\&source=editors\&ust=1684532198672992\&usg=AOvVaw1qZrehsCyrctZGXFgM1f5u) [Step 4: Review details on run setup page](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol%23Step_4\&sa=D\&source=editors\&ust=1684532198673484\&usg=AOvVaw0M5BnVyS7m96S9hMcmYvOG) [Setup](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol%23Setup\&sa=D\&source=editors\&ust=1684532198673959\&usg=AOvVaw08v9Dysk66RRF1gpi-RDTN) [Module Controls](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol%23Module_Controls\&sa=D\&source=editors\&ust=1684532198674460\&usg=AOvVaw0tu2wfI7h4lcG7iDzO3PND) [Run Preview](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol%23Run_Preview\&sa=D\&source=editors\&ust=1684532198675461\&usg=AOvVaw1JTQKUU6d2iDVStTN17p21) [Step 5: Start run](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol%23Step_5\&sa=D\&source=editors\&ust=1684532198676494\&usg=AOvVaw1IfmbGL1HR32IQ_WOYz8gW) [Continue to the next section](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Import-a-protocol%23Continue\&sa=D\&source=editors\&ust=1684532198677520\&usg=AOvVaw0hrnxo-QPCFFSXwaKyc5ug) ### ### Introduction ### Before starting If you haven't yet decided on a protocol method, see [Get started: Choose which protocol method is right for you](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Choose-which-protocol-method-is-right-for-you\&sa=D\&source=editors\&ust=1684532198678842\&usg=AOvVaw1C8YCuz638Cj2IeZioywbX). ### ### Procedure ### Step 1: Import Python (.py) or Protocol Designer (.json) file In the Opentrons App, select the Protocols tab. If you have not imported any protocols, you will be able to select Choose File or drag and drop to import your Python (.py) or Protocol Designer (.json) file. ![import protocol\_edit.png](/files/sDtsRKKvpuqXhe5LRAMK) If you have already imported a protocol, this is also where you will see it listed. #### #### Troubleshooting: “Module not found” or “File not found” errors during protocol analysis When a protocol is imported into the app, it undergoes protocol analysis local to the Opentrons App (this is before it is simulated on the OT-2 itself). This process extracts information from the protocol and loads it into the app to display the metadata and graphics. If you are using the Python API and see an error such as “Module not found” for Python packages or “File not found” for csv files loaded onto your OT-2, please review the troubleshooting steps found in [Protocol analysis](https://www.google.com/url?q=https://support.opentrons.com/s/article/Protocol-analysis\&sa=D\&source=editors\&ust=1684532198681108\&usg=AOvVaw1fFlCh7tIcOqd2dW-KIu1e). ### ### Step 2: Select protocol To proceed, select the protocol you want to run. ![first protocol\_edit.png](/files/3AXX6FUKMGwLIQhS9yYh) Note: You can select Import to add more protocols. You can also change the way the list is organized by selecting a different option from the Sort by dropdown menu in the upper right. ### ### Step 3: Proceed to run setup After selecting your protocol, you'll be taken to the protocol overview page. Select Run protocol to continue setting up your run. ![protocol page\_edit.png](/files/pWLGgnZQZb6nPi6AqHwF) You will be prompted to choose an available robot before you can select Proceed to setup. ![choose robot\_edit.png](/files/X6eO6tVRtv4pb7b8WaOJ) #### #### Troubleshooting: Help! I don't see any available robots listed / I'm unable to connect If this is your first time connecting to the robot, please see: * [Get started: Connect to your OT-2 over USB](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Connect-to-your-OT-2-over-USB\&sa=D\&source=editors\&ust=1684532198683707\&usg=AOvVaw2gkxUEpBxqjyC4UVyD9Nbx) * [Get started: Connect to your OT-2 over Wi-Fi (optional)](https://www.google.com/url?q=https://support.opentrons.com/s/article/Get-started-Connect-to-your-OT-2-over-Wi-Fi-optional\&sa=D\&source=editors\&ust=1684532198684287\&usg=AOvVaw0HpjTjyZCsx1S34KFbxCwt) If you've successfully connected to the robot before but are suddenly unable to, please see: * [Troubleshooting problems with USB connections](https://www.google.com/url?q=https://support.opentrons.com/s/article/Troubleshooting-connection-problems\&sa=D\&source=editors\&ust=1684532198685021\&usg=AOvVaw3tbEWY5XjfPKqWWeU7CEXv) * [Troubleshooting problems with Wi-Fi connections](https://www.google.com/url?q=https://support.opentrons.com/s/article/Troubleshooting-problems-with-Wi-Fi-connections\&sa=D\&source=editors\&ust=1684532198685591\&usg=AOvVaw3KllK-kxy-M-tWmJENgIK4) ### ### Step 4: Review details on run setup page Details about your protocol are broken down into 3 tabs on the run setup page: * Setup * Module Controls * Run Preview #### #### Setup The Setup tab displays the statuses of Robot Calibration, Module Setup (if your protocol uses Opentrons modules), Labware Setup, and Liquid Setup. Click on a step to expand it. ![setup run\_edit.PNG](/files/g2wKw3WTyGVTomLum41P) Note: If your protocol does not use any modules, the Module Setup step will not be shown. Robot Calibration allows you to review current calibration data and will flag any required calibrations that are missing or need to be redone. ![setup run\_cals\_edit.PNG](/files/HNyAPat6m1TEsiEsjGW5) Module Setup displays information about the modules used in the protocol. ![setup run\_mods\_edit.PNG](/files/MMQPdbV3LNSzloheuWvk) Labware Setup displays information about labware used in the protocol. This is also where you can run Labware Position Check. See [How Labware Offsets work](https://www.google.com/url?q=https://support.opentrons.com/s/article/How-Labware-Offsets-work-on-the-OT-2\&sa=D\&source=editors\&ust=1684532198689551\&usg=AOvVaw1G74g86d-hPbaH2B84I35E) for additional details. ![setup run\_labware\_edit.PNG](/files/RG3MEBeYPHzulSyuSQls) Initial Liquid Setup displays the volume and starting location of each liquid used in the protocol. Click on a liquid to further expand it. ![setup run\_liquid\_expand\_edit.PNG](/files/iyv7QyVrcoxyo3GYeHyu) You can click on an expanded liquid's details to see specific information about the labware containing the liquid, as well as its starting deck state. ![setup run\_liquid\_expand\_select.PNG](/files/JEFimfaSMdik0HBRUMcT) ![setup run\_liquid\_detail\_edit.PNG](/files/5P0eyFNwnQIE0xP2eK7W) #### #### Module Controls The Module Controls tab allows you to interact with connected Opentrons modules that will be used in your protocol. The protocol in the example shown throughout this article uses a Magnetic Module. You can set the engage height of the magnets by selecting the three-dot menu (⋮) > Set engage height. ![setup run\_mod controls\_three-dot\_edit.PNG](/files/L6CsHYPVqwPXWWTt899U) #### #### Run Preview The Run Preview tab displays a preview of your protocol's steps. ![setup run\_run preview\_edit.png](/files/Z1VN8KtCZLpItfJtSf7M) ### ### Step 5: Start run When you're ready to run your protocol, select Start run at the top. ![setup run\_start.PNG](/files/guvDpNuGYKTx3lnLn0nJ) Copyright © 2023 Opentrons # More Parheliabio.com content # Howitworks [Skip to content](https://www.google.com/url?q=https://parheliabio.com/how-it-works/%23content\&sa=D\&source=editors\&ust=1684533011297156\&usg=AOvVaw36dJwP1vn0A2jR-CDsla4f) * [MY ACCOUNT](https://www.google.com/url?q=https://parheliabio.com/my-account/\&sa=D\&source=editors\&ust=1684533011297482\&usg=AOvVaw3hRybSwGpunnZUVQEzfejo) * [HOME](https://www.google.com/url?q=https://parheliabio.com/\&sa=D\&source=editors\&ust=1684533011297883\&usg=AOvVaw1fgP9pwEyS64aFx72Ey5-1) * [PRODUCTS](https://www.google.com/url?q=https://parheliabio.com/shop/\&sa=D\&source=editors\&ust=1684533011298143\&usg=AOvVaw2Qkm6yK108sc4wOaYfxAu2) * [HOW IT WORKS](https://www.google.com/url?q=https://parheliabio.com/how-it-works/\&sa=D\&source=editors\&ust=1684533011298489\&usg=AOvVaw39lbrv1rkajkbIhvQ6Ms3p) * [SUPPORT & RESOURCES](https://www.google.com/url?q=https://parheliabio.com/support/\&sa=D\&source=editors\&ust=1684533011298839\&usg=AOvVaw3Ccrkx65PNrBcWh74HptFo) * [ABOUT US](https://www.google.com/url?q=https://parheliabio.com/about-us/\&sa=D\&source=editors\&ust=1684533011299164\&usg=AOvVaw3RTT6_NV3YZbB1lz5g4AWd) * [CONTACT](https://www.google.com/url?q=https://parheliabio.com/contact/\&sa=D\&source=editors\&ust=1684533011299430\&usg=AOvVaw3TkPi1BcI6dQiKHvmLZw3u) * [BLOG](https://www.google.com/url?q=https://parheliabio.com/blog/\&sa=D\&source=editors\&ust=1684533011299714\&usg=AOvVaw0gM9BCX2YwIxudX19ulaEh) * [STAINWORKS™](https://www.google.com/url?q=https://3qcwn6iy48.execute-api.us-east-1.amazonaws.com/\&sa=D\&source=editors\&ust=1684533011300017\&usg=AOvVaw2WxBRELMpc26hQeOufQVcf) **PRECISION TISSUE STAINING EVERY TIME** **AT THE PUSH OF A BUTTON** ### How it works Greater workflow efficiency - saves time and reduces the need for dedicated personnel and training - automated liquid handlers are faster than manual pipetting. [COVERSLIPS](https://www.google.com/url?q=https://parheliabio.com/how-it-works/%23coverslips\&sa=D\&source=editors\&ust=1684533011300861\&usg=AOvVaw1l62RJuBR8-px6vNyLrF6P)[ ](https://www.google.com/url?q=https://parheliabio.com/how-it-works/%23coverslips\&sa=D\&source=editors\&ust=1684533011301135\&usg=AOvVaw1SHVs9s-C3nJkI8QqThAPO) [SLIDES](https://www.google.com/url?q=https://parheliabio.com/how-it-works/%23slides\&sa=D\&source=editors\&ust=1684533011301409\&usg=AOvVaw17QkusoeXeBaCJxJAzjlZL)[ ](https://www.google.com/url?q=https://parheliabio.com/how-it-works/%23slides\&sa=D\&source=editors\&ust=1684533011301624\&usg=AOvVaw2aLlkaJxbjw_xNL0Vpu_j5) [MANUAL STAINING](https://www.google.com/url?q=https://parheliabio.com/how-it-works/%23manual\&sa=D\&source=editors\&ust=1684533011301876\&usg=AOvVaw2dMoWgkUp4_309s2YZYWzv)[ ](https://www.google.com/url?q=https://parheliabio.com/how-it-works/%23manual\&sa=D\&source=editors\&ust=1684533011302160\&usg=AOvVaw3sVrxeCEY3rTh1ooRg6qlw) [STAINING AUTOMATION](https://www.google.com/url?q=https://parheliabio.com/how-it-works/%23automation\&sa=D\&source=editors\&ust=1684533011302446\&usg=AOvVaw07mAs9xsbXCOpwXh5C7cRj)[ ](https://www.google.com/url?q=https://parheliabio.com/how-it-works/%23automation\&sa=D\&source=editors\&ust=1684533011302665\&usg=AOvVaw1lyqCfC_Lbx-LVb4FRhkqh) [HEATING & COOLING ON THE ROBOT](https://www.google.com/url?q=https://parheliabio.com/how-it-works/%23heating-cooling\&sa=D\&source=editors\&ust=1684533011302921\&usg=AOvVaw3-LimF5G46j3N-qmJOxIXS)[ ](https://www.google.com/url?q=https://parheliabio.com/how-it-works/%23heating-cooling\&sa=D\&source=editors\&ust=1684533011303141\&usg=AOvVaw2Exd7wcD_yVuid7SomQyNv) [LIQUID HANDLING](https://www.google.com/url?q=https://parheliabio.com/how-it-works/%23liquid-handling\&sa=D\&source=editors\&ust=1684533011303417\&usg=AOvVaw3A5RHWbSjdaLLlx-dMPHG3)[ ](https://www.google.com/url?q=https://parheliabio.com/how-it-works/%23liquid-handling\&sa=D\&source=editors\&ust=1684533011303632\&usg=AOvVaw2TN-4OMj9l3-9TPcbG9Yk-) **MAKING TISSUE AUTO-STAINING ACCESSIBLE TO ALL** ### Peace of mind of having reproducible results – with simple or complex protocols ### Omni-Stainer™ C12 for coverslips * Compact: footprint of a 96-well plate * Stains up to 12 coverslip-mounted samples at a time * Saves reagents: 50% less reagents compared to open-top staining * Never lose or break a sample * Built-in wet chamber * Use it by hand or insert into any SBS-compatible liquid handler to automate your staining ![Omni-stainer-C12-with-tip-open-lid-3](/files/Amc6oM0J8YRfSNeMx5h5) [VIEW PRODUCTS](https://www.google.com/url?q=https://parheliabio.com/shop/\&sa=D\&source=editors\&ust=1684533011305063\&usg=AOvVaw0lpxGE7has4GuenKquH7lx) [FAQ - GET ANSWERS](https://www.google.com/url?q=https://parheliabio.com/frequently-asked-questions/\&sa=D\&source=editors\&ust=1684533011305332\&usg=AOvVaw0ZuZHS6Q82-T7kci8Ehxyt) ### How it works for coverslips ![how-it-works-coverslips](/files/vkABFgO5xHFMYOiKxSER) 1. Coverslip with sample rests on 50um thin spacers, forming a capillary chamber. Staining reagents are supplied by a liquid handler (or by hand!) 2. Sample is incubated in a thin capillary layer 3. When the sample is washed, the old one is instantly displaced by the laminar flow ### Omni-Stainer™ S12 for slides * Compact: footprint of a 96-well plate * Stains up to 12 coverslip-mounted samples at a time * Saves reagents: 50% less reagents compared to open-top staining * Never lose or break a sample * Built-in wet chamber * Use it by hand or insert into any SBS-compatible liquid handler to automate your staining [VIEW PRODUCTS](https://www.google.com/url?q=https://parheliabio.com/shop/\&sa=D\&source=editors\&ust=1684533011307074\&usg=AOvVaw2rX8P3DIbpQ_LWyEepQwET) [FAQ - GET ANSWERS](https://www.google.com/url?q=https://parheliabio.com/frequently-asked-questions/\&sa=D\&source=editors\&ust=1684533011307353\&usg=AOvVaw14ZD5EIuvsJ64BZnBvlEu6) ### How it works for slides ![how-it-works-for-slides](/files/vBHOahFtZslp0WQF8GOk) * The same capillary displacement principle, but the sample is mounted on a slide and covered with a coverpad that incorporates capillary spacers **EFFICIENT - ACCURATE - AFFORDABLE** #### View our virtual demo ![Virtual Demo](/files/cHTgyTcVpYlLyC1o460D) Complete your details and view our virtual demonstration of the Omni-Stainer™ Name(Required) Email(Required) Organisation(Required) ### Placing slides into Omni-Stainer™ Slide assembly\ This video shows how to assemble the slide/coverpad sandwich and place it into the slide Omni-StainerTM. First the tacky spacers are exposed by removing the protective tape from the cover pad. To avoid airbubbles between the slide and the coverpad the slide is first placed in the small tray with compatible buffer (water or PBS) and in such submerged stage is covered with a coverpad. The spacers are tacky enough to hold the coverpad in place (after slightly pressed against the slide) but yet when wet allow repositioning the coverpad over the slide. After the sandwich is assembled the combo is placed on the shelf and then into the Omni-StainerTM Play Video Coverslip Assembly\ This video shows how to assemble the coverslip/support pad sandwich and place it into the coverslip Omni-StainerTM system. To avoid airbubbles between the slide and the support pad, a drop of compatible buffer (water or PBS) is first placed onto the support pad. The coverslip with a sample is then placed face down on the pad to avoid bubble formation and ensure that the liquid fully fills the flow cell formed by sandwiching the support pad and the coverslip. After the sandwich is assembled the combo is placed into the coverslip version of the Omni-StainerTM system. Play Video ### Manual staining Using Omni-Stainer™ platform by hand Play Video Omni-Stainer™ S12 platform Demo Video Play Video Omni-Stainer™ C12 platform Demo Video The Omni-StainerTM platform can be used as a device for manual staining. In such case the solutions are exchanged by manual pipetting. After the pipetting the Omni-StainerTM system should be closed with a lid. In such configuration it can be placed into the 4C refrigerator or into the heated incubator. Before the start of any staining experiment 20-30ml of water should be placed into the Omni-StainerTM system to create the humid chamber environment which helps to reduce the evaporation of staining solutions from the slide/coverpad or coverslip/support pad sandwich.” [VIEW PRODUCTS](https://www.google.com/url?q=https://parheliabio.com/shop/\&sa=D\&source=editors\&ust=1684533011310846\&usg=AOvVaw0OiaNfAxqOaaUJVas2IW5y) [VIEW SAMPLE DATA](https://www.google.com/url?q=https://parheliabio.com/sample-data/\&sa=D\&source=editors\&ust=1684533011311133\&usg=AOvVaw0J2Ms_DXJYXm-TFfP_VTWp) ### ### Automated Staining Demo using Opentrons OT-2 {% embed url="" %} Omni-Stainer™ S12 platform {% endembed %} Playback speed increased^ Omni-Stainer™ S12 platform automation demo {% embed url="" %} Omni-Stainer™ C12 platform Demo Video For automated liquid exchange the Omni-Stainer™ system has to be place into programmable liquid handler (e.g. Opentrons). The design of the lid including the prewetted perforated filter paper on the lid ensures minimizing the evaporation of staining reagents from the slide/coverpad or coverslip/support pad sandwich. [VIEW PRODUCTS](https://www.google.com/url?q=https://parheliabio.com/shop/\&sa=D\&source=editors\&ust=1684533011312020\&usg=AOvVaw0jgdSbicMcmRrU3221ybSG) [VIEW SAMPLE DATA](https://www.google.com/url?q=https://parheliabio.com/sample-data/\&sa=D\&source=editors\&ust=1684533011312273\&usg=AOvVaw2KSnrr8kzjzBdqZ1SwHXMK) ### Liquid handling OT-2 Staining automation can be attained by inserting the Omni-Stainer™ into an SBS-compatible liquid handler, and how we are partnering with Opentrons and adopting their versatile ultra-low-cost liquid robot OT-2 to deliver complete staining automation solutions. As a part of our ‘[automation package](https://www.google.com/url?q=https://parheliabio.com/product/custom-protocol-development/\&sa=D\&source=editors\&ust=1684533011313031\&usg=AOvVaw1WY8XC-FOue4S4s-c89gyZ)’, we provide pre-written protocols for various assays, including H\&E, IHC, IF, CycIF, CODEX, FFPE antigen retrieval, CAS-FISH, IMC, RNAscope. [Custom protocol development services are available.](https://www.google.com/url?q=https://parheliabio.com/product/custom-protocol-development/\&sa=D\&source=editors\&ust=1684533011313294\&usg=AOvVaw3QKr9NtuHAwvz3OMKvTP8A) **Omni-Stainer™ is compatible with most liquid handling robots but if you don't have one, we recommend Opentrons OT-2** Protocols Supported on OT-2: * IF * IHC * CODEX single and multi-strain * Cas-FISH * FFPE Antigen Retrieval (HIER) * Opal Coming soon: * smRNA-FISH * Rolling Circle Amplification * Cyclic IF * IMC & MIBI * and many more... ![OT 2](/files/ptPtZO7rbAsPpCysud0W) ### Heating and cooling on the robot: FFPE-IHC Omni-Stainer™ system rests on top of the OT-2 thermal module after:\ Human tonsil FFPE antigen retrieval at 95°C\ Anti-CD8 Primary antibody staining at 4°C\ Secondary antibody staining, DAB and hematoxylin at room temperature [VIEW PRODUCTS](https://www.google.com/url?q=https://parheliabio.com/shop/\&sa=D\&source=editors\&ust=1684533011315283\&usg=AOvVaw3dYumHk6X-QYaXYvOsD2LI) [FAQ - GET ANSWERS](https://www.google.com/url?q=https://parheliabio.com/frequently-asked-questions/\&sa=D\&source=editors\&ust=1684533011315557\&usg=AOvVaw0O8DyAhYo1CRlrew4y4ht7) ![Heating and cooling on the robot: FFPE-IHC](/files/X8WdzGSnKmaa7vqQlEov) #### Are you ready for the Omni-Stainer™ platform to transform your life? [VIEW PRODUCTS](https://www.google.com/url?q=https://parheliabio.com/shop/\&sa=D\&source=editors\&ust=1684533011316030\&usg=AOvVaw04kWjTfknZGGjssr2JohUh) © 2023 Parhelia Biosciences | [Website Privacy Policy](https://www.google.com/url?q=https://parheliabio.com/privacy-policy/\&sa=D\&source=editors\&ust=1684533011316401\&usg=AOvVaw3pXOczuqOQDrCb2gKPwE9C) |[ Terms and Conditions](https://www.google.com/url?q=https://parheliabio.com/terms-conditions\&sa=D\&source=editors\&ust=1684533011316640\&usg=AOvVaw1IaRfbWvT6C9Wr4Inkcl38) | [Cookie Policy](https://www.google.com/url?q=https://parheliabio.com/cookie-policy\&sa=D\&source=editors\&ust=1684533011316867\&usg=AOvVaw2QdYJEa3wR9OPzt1GyGDMS) |[ Website by Arttia Creative](https://www.google.com/url?q=https://arttia.co.uk/\&sa=D\&source=editors\&ust=1684533011317092\&usg=AOvVaw26q2v6xNYZ6zXXIi4gk1On) # Parhelia Biosciences FAQs How does Omni-Stainer™ work? #### How does Omni-Stainer™ work? Omni-Stainer™ enables microscopy staining automation by hosting samples in thin capillary exchange chambers, thus allowing a liquid handler to supply reagents to the tops of the chambers. When a new buffer is added to the top, it is drawn into the chamber by the capillary force, instantly displacing the old buffer. Capillary wash of a Cover Pad-mounted sample in Omni-Stainer™ C12 What sample types does the Omni-Stainer™ support? What coverslip types does Omni-Stainer™ support? What types of applications require coverslip staining? What types of applications require slide-mounted samples? Does Omni-Stainer™ support heating and cooling? Which liquid handlers are compatible with the Omni-stainer device? How does Omni-Stainer™ compare to other autostaining solutions? What kind of buffers does Omni-Stainer™ support? # Page 1 # Frequently asked questions parheliabio website **Frequently asked questions** **Frequently asked questions** How does Omni-Stainer™ work? **How does Omni-Stainer™ work?** Omni-Stainer™ enables microscopy staining automation by hosting samples in thin capillary exchange chambers, thus allowing a liquid handler to supply reagents to the tops of the chambers. When a new buffer is added to the top, it is drawn into the chamber by the capillary force, instantly displacing the old buffer. **What sample types does the Omni-Stainer™ support?** Omni-Stainer™ exists in two versions: Omni-Stainer™ S12 for slide-mounted samples and Omni-Stainer™ C12 for coverslip-mounted samples. Please make sure to order the version according to your needs. **What coverslip types does Omni-Stainer™ support?** The coverslip instrument Omni-Stainer™ can accommodate samples mounted standard rectangular microscopy coverslips, 22x22 or 18x18 size. **What types of applications require coverslip staining?** Coverslip staining is less conventional than staining on slides but it is widely used in a number of applications, such as: 1. Cell cultures - adherent cell lines are often grown on coverslips as they easily adhere to the glass and form micro-colonies 2. TIRF - Total Internal Reflection Microscopy illuminates a thin surface layer of approx. \~200nm and therefore requires the sample to be adhered directly to the coverslip 3. Cyclic protocols that require on-microscope sample perfusion protocols, such as CODEX, CycIF, MERFISH, variations of STORM such as Exchange-PAINT - all require coverslip-mounted samples. **What types of applications require slide-mounted samples?** Most OCT fresh-frozen or FFPE tissue sample staining protocols are performed on slides. Those include Immunohistochemistry (IHC), Immunofluorescence (IF), In Situ Hybridization (ISH), Imaging Mass Cytometry (IMC). **Does Omni-Stainer™ support heating and cooling?** The Omni-Stainer™ is made of heat-conductive aluminum and can be used in conjuncture with a temperature control module (like the one here), allowing the temperature to be programmatically controlled between 4C and 95C. This enables a variety of protocols, such as FFPE Antigen Retrieval, smRNA-FISH, or CycIF to be performed directly inside the Omni-Stainer™ device. **Does Omni-Stainer™ support heating and cooling?** The Omni-Stainer™ is made of heat-conductive aluminum and can be used in conjuncture with a temperature control module (like the one here), allowing the temperature to be programmatically controlled between 4C and 95C. This enables a variety of protocols, such as FFPE Antigen Retrieval, smRNA-FISH, or CycIF to be performed directly inside the Omni-Stainer™ device. smRNA-FISH **Which liquid handlers are compatible with the Omni-Stainer™ device?** Omni-Stainer™ can be used in most liquid handling robots that support 96-well-plate-sized labware, including Hamilton, Agilent Bravo, Tecan Evo, and others. For those labs that would need to purchase a liquid handler to enable Parhelia automation, we recommend Opentrons OT-2, a simple budget liquid handler that is available [here](https://parheliabio.com/product-category/opentrons-robot-purchase/) ![Automated Staining System](/files/W8Q5PCQaq3TaI3XV1jtr) Omni-Stainer™ inside a liquid handling robot **How does Omni-Stainer™ compare to other autostaining solutions?** There are many staining automation solutions on the market, with big names from prominent vendors. However, most of those systems are bulky, expensive, not particularly reagent-efficient, and are oriented toward clinical pathology labs. Since they are not designed to be used in a research setting, such systems can be difficult to program and adapt to custom protocols. In contrast, Omni-Stainer™ is compact, affordable, extremely flexible and easy to use. In addition, the Omni-Stainer™ has the unique capability to stain coverslip-mounted samples. ![Parhelia Omni-Stainer](/files/8O2nb3zoi9koN1obTrKD) Omni-Stainer™ hosts 12 coverslips samples and 12 slide samples. **What kind of buffers does Omni-Stainer™ support?** Omni-Stainer™ is made of chemically inert materials and is compatible with most aqueous buffers including those containing high-viscosity agents (e.g. dextran sulfate, glycerol), high salt buffers (up to 0.65M salt), detergents, DMF/DMSO, semi-polar organic solvents (methanol, ethanol, isopropanol, acetone). It is not compatible with non-polar solvents such as petrol distillates or xylenes. !\[A picture containing diagram, plan, map Description automatically generated]\(/files/AoxG7AjmDOLghrZOdulm) Dextran sulfate # ParheliaFrequentlyaskedquestions Frequently asked questions Frequently asked questions How does Omni-Stainer™ work? How does Omni-Stainer™ work? Omni-Stainer™ enables microscopy staining automation by hosting samples in thin capillary exchange chambers, thus allowing a liquid handler to supply reagents to the tops of the chambers. When a new buffer is added to the top, it is drawn into the chamber by the capillary force, instantly displacing the old buffer. What sample types does the Omni-Stainer™ support? Omni-Stainer™ exists in two versions: Omni-Stainer™ S12 for slide-mounted samples and Omni-Stainer™ C12 for coverslip-mounted samples. Please make sure to order the version according to your needs. What coverslip types does Omni-Stainer™ support? The coverslip instrument Omni-Stainer™ can accommodate samples mounted standard rectangular microscopy coverslips, 22x22 or 18x18 size. What types of applications require coverslip staining? Coverslip staining is less conventional than staining on slides but it is widely used in a number of applications, such as: 1\. Cell cultures - adherent cell lines are often grown on coverslips as they easily adhere to the glass and form micro-colonies 2\. TIRF - Total Internal Reflection Microscopy illuminates a thin surface layer of approx. \~200nm and therefore requires the sample to be adhered directly to the coverslip 3\. Cyclic protocols that require on-microscope sample perfusion protocols, such as CODEX, CycIF, MERFISH, variations of STORM such as Exchange-PAINT - all require coverslip-mounted samples. What types of applications require slide-mounted samples? Most OCT fresh-frozen or FFPE tissue sample staining protocols are performed on slides. Those include Immunohistochemistry (IHC), Immunofluorescence (IF), In Situ Hybridization (ISH), Imaging Mass Cytometry (IMC). Does Omni-Stainer™ support heating and cooling? The Omni-Stainer™ is made of heat-conductive aluminum and can be used in conjuncture with a temperature control module (like the one here), allowing the temperature to be programmatically controlled between 4C and 95C. This enables a variety of protocols, such as FFPE Antigen Retrieval, smRNA-FISH, or CycIF to be performed directly inside the Omni-Stainer™ device. Does Omni-Stainer™ support heating and cooling? The Omni-Stainer™ is made of heat-conductive aluminum and can be used in conjuncture with a temperature control module (like the one here), allowing the temperature to be programmatically controlled between 4C and 95C. This enables a variety of protocols, such as FFPE Antigen Retrieval, smRNA-FISH, or CycIF to be performed directly inside the Omni-Stainer™ device. smRNA-FISH Which liquid handlers are compatible with the Omni-Stainer™ device? Omni-Stainer™ can be used in most liquid handling robots that support 96-well-plate-sized labware, including Hamilton, Agilent Bravo, Tecan Evo, and others. For those labs that would need to purchase a liquid handler to enable Parhelia automation, we recommend Opentrons OT-2, a simple budget liquid handler that is available [here](https://www.google.com/url?q=https://parheliabio.com/product-category/opentrons-robot-purchase/\&sa=D\&source=editors\&ust=1684535517200253\&usg=AOvVaw2ytiaZgDaudtE3T4f4iQO7) Omni-Stainer™ inside a liquid handling robot How does Omni-Stainer™ compare to other autostaining solutions? There are many staining automation solutions on the market, with big names from prominent vendors. However, most of those systems are bulky, expensive, not particularly reagent-efficient, and are oriented toward clinical pathology labs. Since they are not designed to be used in a research setting, such systems can be difficult to program and adapt to custom protocols. In contrast, Omni-Stainer™ is compact, affordable, extremely flexible and easy to use. In addition, the Omni-Stainer™ has the unique capability to stain coverslip-mounted samples. Omni-Stainer™ hosts 12 coverslips samples and 12 slide samples. What kind of buffers does Omni-Stainer™ support? Omni-Stainer™ is made of chemically inert materials and is compatible with most aqueous buffers including those containing high-viscosity agents (e.g. dextran sulfate, glycerol), high salt buffers (up to 0.65M salt), detergents, DMF/DMSO, semi-polar organic solvents (methanol, ethanol, isopropanol, acetone). It is not compatible with non-polar solvents such as petrol distillates or xylenes. Dextran sulfate # Parhelia Omni-Stainer FAQ Frequently asked questions How does Omni-Stainer™ work? Omni-Stainer™ enables microscopy staining automation by hosting samples in thin capillary exchange chambers, thus allowing a liquid handler to supply reagents to the tops of the chambers. When a new buffer is added to the top, it is drawn into the chamber by the capillary force, instantly displacing the old buffer. What sample types does the Omni-Stainer™ support? Omni-Stainer™ exists in two versions: Omni-Stainer™ S12 for slide-mounted samples and Omni-Stainer™ C12 for coverslip-mounted samples. Please make sure to order the version according to your needs. What coverslip types does Omni-Stainer™ support? The coverslip instrument Omni-Stainer™ can accommodate samples mounted standard rectangular microscopy coverslips, 22x22 or 18x18 size. What types of applications require coverslip staining? Coverslip staining is less conventional than staining on slides but it is widely used in a number of applications, such as: 1\. Cell cultures - adherent cell lines are often grown on coverslips as they easily adhere to the glass and form micro-colonies 2\. TIRF - Total Internal Reflection Microscopy illuminates a thin surface layer of approx. \~200nm and therefore requires the sample to be adhered directly to the coverslip 3\. Cyclic protocols that require on-microscope sample perfusion protocols, such as CODEX, CycIF, MERFISH, variations of STORM such as Exchange-PAINT - all require coverslip-mounted samples. What types of applications require slide-mounted samples? Most OCT fresh-frozen or FFPE tissue sample staining protocols are performed on slides. Those include Immunohistochemistry (IHC), Immunofluorescence (IF), In Situ Hybridization (ISH), Imaging Mass Cytometry (IMC). Does Omni-Stainer™ support heating and cooling? The Omni-Stainer™ is made of heat-conductive aluminum and can be used in conjuncture with a temperature control module (like the one here), allowing the temperature to be programmatically controlled between 4C and 95C. This enables a variety of protocols, such as FFPE Antigen Retrieval, smRNA-FISH, or CycIF to be performed directly inside the Omni-Stainer™ device. Does Omni-Stainer™ support heating and cooling? The Omni-Stainer™ is made of heat-conductive aluminum and can be used in conjuncture with a temperature control module (like the one here), allowing the temperature to be programmatically controlled between 4C and 95C. This enables a variety of protocols, such as FFPE Antigen Retrieval, smRNA-FISH, or CycIF to be performed directly inside the Omni-Stainer™ device. smRNA-FISH Which liquid handlers are compatible with the Omni-Stainer™ device? Omni-Stainer™ can be used in most liquid handling robots that support 96-well-plate-sized labware, including Hamilton, Agilent Bravo, Tecan Evo, and others. For those labs that would need to purchase a liquid handler to enable Parhelia automation, we recommend Opentrons OT-2, a simple budget liquid handler that is available [here](https://parheliabio.com/product-category/opentrons-robot-purchase/) Omni-Stainer™ inside a liquid handling robot How does Omni-Stainer™ compare to other autostaining solutions? There are many staining automation solutions on the market, with big names from prominent vendors. However, most of those systems are bulky, expensive, not particularly reagent-efficient, and are oriented toward clinical pathology labs. Since they are not designed to be used in a research setting, such systems can be difficult to program and adapt to custom protocols. In contrast, Omni-Stainer™ is compact, affordable, extremely flexible and easy to use. In addition, the Omni-Stainer™ has the unique capability to stain coverslip-mounted samples. Omni-Stainer™ hosts 12 coverslips samples and 12 slide samples. What kind of buffers does Omni-Stainer™ support? Omni-Stainer™ is made of chemically inert materials and is compatible with most aqueous buffers including those containing high-viscosity agents (e.g. dextran sulfate, glycerol), high salt buffers (up to 0.65M salt), detergents, DMF/DMSO, semi-polar organic solvents (methanol, ethanol, isopropanol, acetone). It is not compatible with non-polar solvents such as petrol distillates or xylenes. Dextran sulfate
# Meet your Parhelia Omnistainer This page uses some old nomenclature, such as Cover Tile instead of Cover Pad and Support Tile instead of Support Pad ![](/files/GraA3M7ZOX9mhinxlPXs) Dear Customer, Congratulations on the acquisition of Parhelia Omni-Stainer and welcome to the Parhelia community! While getting familiar with an entirely new system for staining can seem daunting, we are here to help you through every step. This guide will take you through how to use Parhelia Omni-stainer as a standalone instrument as well as in conjunction with protocol automation on Opentrons OT-2. ### What’s inside Parhelia Omni-stainer starter kit #### Overview and assembly instructions [in this video](https://www.google.com/url?q=https://vimeo.com/782691867\&sa=D\&source=editors\&ust=1684536753660330\&usg=AOvVaw1hpXF2_-8cGlsHSTsFxPTL) * Omni-stainer C12/S12: your brand-new instrument precision-machined out of aluminum, anodized for durability and chemical resistance * (S12 only): a set of 4 shelves are designed to be inserted into the Omni-stainer S12 and support standard 25x75mm microscopy slides. * Perforated lid for automation (automation bundle) or Solid lid (manual bundle) * Cover Tiles (S12) or Support Tiles (C12) (starter pack, 10 pcs): single-use staining chambers made of temperature-resistant polycarbonate * Perforated filter paper (starter pack, 3 pcs): 3mm thick filter paper that is laminated to a double-sided adhesive and protected with a liner. The paper needs to be adhered to the underside of the perforated lid and pre-wetted before each run. Its purpose is to create a moisture barrier around the sample access holes and thus prevent sample evaporation during long incubations. * Conditioning reagent - 5ml bottle. Parhelia conditioning reagents is a proprietary mix that optimizes the liquid flow. We recommend adding it at 1:100 to every buffer used in the protocol in order to ensure smooth liquid exchange and reduce bubble formation. We noticed however that if it is added at the rehydration step (when a dry tissue sample is first exposed to an aqueous buffer), it may negatively affect the staining results, so we recommend skipping it during the rehydration step, but using at all other steps that involve aqueous buffers. * Thermal sheath. (If ordered, it will be shipped separately). Thermal Sheath is designed to be used in combination with [OT-2 Temperature Module](https://www.google.com/url?q=https://parheliabio.com/product/opentrons-temperature-module/\&sa=D\&source=editors\&ust=1684536753662076\&usg=AOvVaw3Pm86U45NRUeGZT0NRS-hQ) and insulate Omni-stainer from the top and the sides while it’s being heated from beneath. A sliding shutter on the Thermal Sheath has a tab that is intended to be operated by the tip of the pipette, opening and closing the sample access wells as needed. ### Additional materials needed for the first runs * 12-trough reservoir ([Corning-Axygen](https://www.google.com/url?q=https://ecatalog.corning.com/life-sciences/b2c/US/en/Genomics-%26-Molecular-Biology/Automation-Consumables/Automation-Reservoirs/Axygen%25C2%25AE-Reagent-Reservoirs/p/RES-MW12-HP\&sa=D\&source=editors\&ust=1684536753663180\&usg=AOvVaw2wO7zm8xC_hIksTKMo2m-4)): for large reagents, hosts 12 separate reagents up to 21 mL each * Skirted 96-well PCR plate: for small reagents (antibodies, blockers, dyes, fixatives) * V-shaped polystyrene reservoir for sample loading ([Corning](https://www.google.com/url?q=https://www.fishersci.com/shop/products/costar-sterile-disposable-reagent-reservoirs-1/07200127\&sa=D\&source=editors\&ust=1684536753664041\&usg=AOvVaw1O6R4lQrM7gry5JTw2-gl_)) * Pierceable aluminum foil ([Corning-Axygen](https://www.google.com/url?q=https://ecatalog.corning.com/life-sciences/b2c/US/en/Genomics-%26-Molecular-Biology/PCR-Consumables/Sealing-Films-and-Tapes/Axygen%25C2%25AE-Sealing-Films/p/PCR-AS-200\&sa=D\&source=editors\&ust=1684536753664600\&usg=AOvVaw3xlirm9qWWi7qmdE0CthKv)) * Blue dye demo reagents: * PBS buffer * Blue Dye for demo runs and volume calibration. - Bromophenol blue solution, 0.04% in PBS (dissolve 40mg of Bromophenol blue in 100 mL of PBS and shake until dissolved), or ready-made solution [from Sigma](https://www.google.com/url?q=https://www.sigmaaldrich.com/US/en/product/sial/318744\&sa=D\&source=editors\&ust=1684536753665215\&usg=AOvVaw3cpMlrv5UnvlFlg_kX5FZh) ### Setting up the Opentrons OT-2 (automation bundle only) #### Instrument setup * Follow the setup instructions [on the Opentrons website](https://www.google.com/url?q=https://support.opentrons.com/s/ot2-get-started\&sa=D\&source=editors\&ust=1684536753666157\&usg=AOvVaw1mZ9fzmJf2ht56XJ6OYTRc). You will need: * A computer that will be connected to the OT-2 instrument * An user account with sufficient permissions to install the Opentrons app * A free Ethernet port (or an Ethernet adapter if you use a laptop) #### Import custom labware definitions * Labware definitions are special files in JSON format that contain the information about the geometry of labwares (number of wells, XY positioning, well depth etc) that the Opentrons OT-2 robot relies on to correctly access the samples. We created custom labware definitions that describe Parhelia Omni-stainer instruments and reservoirs (deep-well 12-trough, 96-well black plate and 96-well skirted plate) that we use in our OT-2 protocols.[ Follow this guide](https://www.google.com/url?q=https://parhelia-biosciences.gitbook.io/omni-stainer-user-guide/product-guides/using-the-omni-stainers-in-automatic-liquid-handler-ot-2/importing-labware-definitions\&sa=D\&source=editors\&ust=1684536753667592\&usg=AOvVaw3jXckOWDiwtwBNFmTQp6lx) for labware definitions download and import. #### Protocol programming * Opentrons protocols are written using the Python programming language and make use of Opentrons’ Python API. This innovative feature of the OT-2 makes good use of Python’s combination of simplicity and expressive power, so you can easily program arbitrarily complex protocols even as a beginner. Follow this[ comprehensive guide](https://www.google.com/url?q=https://docs.opentrons.com/v2/writing.html\&sa=D\&source=editors\&ust=1684536753668381\&usg=AOvVaw2hIBhUTXNxNUmE6gcFwKqW) on programming OT-2 with Python. #### StainWorks * Since many researchers are not familiar with with Python, we at Parhelia created StainWorks: a simple yet powerful web app that provides two essential features: * Templating - the ability to configure Python protocols without having to dig into the Python code. Instead, the relevant run parameters (number of samples, wash volumes. * Global Library - a curated collection of Python scripts that implement various assays (CODEX, IHC, H\&E etc). These protocols have been created, validated and maintained by the Parhelia team. * Labware layouts - Each template in StainWorks comes with a special “Labware Layout” spreadsheet that is attached to the template. Labware Layout contains instructions on how to correctly fill reservoirs with reagents and place them onto the deck of an OT-2 robot for a particular protocol run. * Accessing StainWorks requires having a user account. If you purchased an S12 or C12 automation bundle, your StainWorks account will be set up automatically and a 90-day free trial of StainWorks is included with your purchase. You will receive a separate email from with your account credentials. You can learn more about exactly how to use StainWorks in the “Blue Dye Demo” tutorial below. ### Preparing the Parhelia Omni-stainer for the run * Place Parhelia Omni-stainer on the bench * Pour 10mL of ddH2O to the waste tray at the bottom- this will maintain the humidity inside the instrument and prevent sample evaporation. * Automation bundle only: * Place the lid on the bench, with the underside facing up, make sure it’s clean and dry. Take one perforated blotting paper sheet and remove the liner. Carefully position the adhesive side over the underside of the lid, making sure that the holes in the paper and the lids line up and press it down. * Use the ddH2O squirt bottle to infuse the blotting paper with 3-5 mL of water. Don’t overdo it - the paper needs to be wet, but not soaking. * Load the samples following the instructions below ### Sample preparation and loading * Preparing samples * FFPE sections: a sample needs to be baked, deparaffinized with Xylenes or a suitable alternative and re-hydrated. If HIER is needed, at this time we recommend it to be. It is possible to do HIER directly in Parhelia Omni-stainer using the Thermal Sheath, but that functionality is experimental and hasn’t been fully tested. Please reach out to us directly at if you would like to try running HIER with Parhelia Omni-stainer. * Fresh frozen sections: sample needs to be fixed and re-hydrated * Cell culture/cell spreads: samples needs to be fixed and re-hydrated * Slide-mounted samples * Setup: ![](/files/GEHwJHl0zdyDeqiKb4u1) * Take a Cover Tile and remove the peel tabs, [following this video](https://www.google.com/url?q=https://vimeo.com/781376730\&sa=D\&source=editors\&ust=1684536753671582\&usg=AOvVaw0mF-0PqGsXeql8TZnDnpq_) * Mount the cover tile as shown and [follow this video](https://www.google.com/url?q=https://vimeo.com/user151431853\&sa=D\&source=editors\&ust=1684536753672062\&usg=AOvVaw0WnNG8HMK_WXH2XiByFM9Z) * Coverslip-mounted samples: * Setup: ![](/files/tbdFwTWKTYYK1h81WNQO) * Follow[ this video ](https://www.google.com/url?q=https://vimeo.com/781371922\&sa=D\&source=editors\&ust=1684536753672963\&usg=AOvVaw2Nm7dIGI3h0_Vi7ec2VGTV)for mounting coverslip samples onto Support tiles * * Preparing samples * **FFPE sections:** a sample needs to be baked, deparaffinized with Xylenes or a suitable alternative and re-hydrated. If HIER is needed, at this time we recommend it to be. It is possible to do HIER directly in Parhelia Omni-stainer using the Thermal Sheath, but that functionality is experimental and hasn’t been fully tested. Please reach out to us directly at if you would like to try running HIER with Parhelia Omni-stainer. * **Fresh frozen sections:** sample needs to be fixed and re-hydrated * **Cell culture/cell spreads:** samples needs to be fixed and re-hydrated * Ensure that the coverslip is flush with the bottom edge of the support tile and is touching the wicking pillar. * Ensure that there is a capillary coupling between the staining chamber and the pillar (basically, a drop of liquid connecting the two). If that’s not the case, dispense 100ul of PBS mixed with 1ul of Parhelia conditioning reagent onto the contact point between the wicking pillar and the support tile. ### Blue Dye Demo This is a very simple demo protocol that helps you understand how capillary gap exchange works inside Parhelia Omni-stainer and calibrate volumes that are needed for the gap exchange. #### Manual run * Prepare two Eppendorf tubes and fill one with PBST and the other one with Blue Dye (1.0 mL each). Add 10ul of Parhelia Conditioning reagent to each, and mix thoroughly. * Using P200 pipette, slowly dispense 110 ul of Blue Dye onto the top curved edge of the S12 covertiles (for C12 coverslips dispense 60ul the solution onto the ramp). Carefully observe how the Blue Dye is drawn into the chamber. Ensure that the clear liquid that was in the staining chamber gets displaced completely and drips down the wicking pillar. Next, wash the staining chamber by dispensing 150ul of the clear buffer. Ensure that the blue dye gets displaced completely. Watch these videos for reference: * [Omni-Stainer C12](https://www.google.com/url?q=https://vimeo.com/781362881\&sa=D\&source=editors\&ust=1684536753674292\&usg=AOvVaw2PIEXkKostoXPac3YscRTn) Manual demo * [Omni-Stainer S12](https://www.google.com/url?q=https://vimeo.com/751961205\&sa=D\&source=editors\&ust=1684536753674687\&usg=AOvVaw20CuCRxoBYs_Q4AbCoX9pL) Manual demo * Very small pockets of carryover buffer towards the bottom corners of the staining chamber are normal and should not affect staining quality because usually there’s no tissue present there. However, if you wish for a more complete exchange, you may wish to increase the wash volume as necessary. * If the flow seems abnormally slow, and the exchange seems incomplete and uneven: * check for bubbles in the chamber. If bubbles are present, slide the sample up and down to release the bubble. If that doesn’t help, re-mount the sample. * Make sure that the sample is positioned correctly within the staining chamber. For coverslips, make sure that the coverslip rests evenly on both spacers. #### Automated run * Open [StainWorks](https://www.google.com/url?q=https://3qcwn6iy48.execute-api.us-east-1.amazonaws.com/login\&sa=D\&source=editors\&ust=1684536753675825\&usg=AOvVaw0HJKV3rl9Hr-G6dDLD29u-) * Go to “Global Library”, locate “Blue Dye Demo” protocol and copy it to your Templates![](/files/FiwZCyPBEzkHjTb4BXxN) * Download the ‘Blue Dye Demo labware layout.xlsx’ file and follow the instructions inside for filling the labwares, also [watch this video](https://www.google.com/url?q=https://vimeo.com/781369840\&sa=D\&source=editors\&ust=1684536753676460\&usg=AOvVaw2qVHup3c1-HbQYnF1D5QCF). * Place the labwares on the deck according to the Labware Layout, making sure that the recessed corner is in the northwest (top-left) position, also refer to[ this video](https://www.google.com/url?q=https://vimeo.com/781366854\&sa=D\&source=editors\&ust=1684536753676829\&usg=AOvVaw1nmhX8wFYZlNBvxB2nGHAk). * Take a blank microscopy slide (for S12) or coverslip (for C12), wipe it clean and dry and mount it with a cover tile/support tile according to the directions above. Place it into the position #1 (the one closest to the recessed corner) of the Parhelia Omni-stainer. * In StainWorks, press “modify protocol” ![](/files/C6R9lNY6d5LKk7QDT1XF) * In the dialogue that pops up, select the type of your Omni-stainer (C12 or S12), keep all other values default. * Press “Save and Download” * Upload the .py file that you just downloaded into the Opentrons app. * Close the lid of the Omni-stainer. Visually locate the calibration mark, which looks like + sign embossed on the lid (note that for C12 and S12, the marks are in different locations). Proceed to run the protocol calibration with the lid closed, following the instructions [in the video](https://www.google.com/url?q=https://vimeo.com/779408843\&sa=D\&source=editors\&ust=1684536753677834\&usg=AOvVaw0iyVZ6kgKrKyC-fTZdbi_l). * If calibration looks good, proceed with the protocol run. * CAUTION: how to correctly re-run the protocol: * Just clicking “Run again”, then “Start Run” will not apply your saved calibration data and the pipetting locations could be off. * To re-run a previously loaded protocol, select “Protocols” on the left side, choose the protocol, then select Run protocol, choose the OT-2 robot and select “Proceed to Setup”. * On the Setup tab click open "STEP 1 Robot Calibration", click Proceed to labware setup. A pop-up window will appear will the calibration data, click Apply stored data, click “Proceed to Run”. * Click “Start run” * Remove the lid and run the protocol to observe the exchange. This is what is should look like: * Parhelia Omni-stainer S12 [blue dye demo](https://www.google.com/url?q=https://vimeo.com/783167726/877293299d\&sa=D\&source=editors\&ust=1684536753678975\&usg=AOvVaw1jmZHPl142UB5enfJ6eGZw) * Parhelia Omni-stainer C12 [blue dye demo](https://www.google.com/url?q=https://vimeo.com/751961154\&sa=D\&source=editors\&ust=1684536753679366\&usg=AOvVaw2m2DsGaGFtto7mXHq6cLhT) * Ensure that the blue dye filling and washing is complete * Very small pockets of carryover buffer towards the bottom corners of the staining chamber are normal and should not affect staining quality because usually there’s no tissue present there. However, if you wish for a more complete exchange, you may wish to increase the wash volume as necessary. * If the flow seems abnormally slow, and the exchange seems incomplete and uneven: * * Carefully check for bubbles in the chamber. Bubbles may form if the buffers used have significant amounts of dissolved gas.If bubbles are present, slide the sample up and down to release the bubble. If that doesn’t help, re-mount the sample. When in doubt, degas buffers before the run. * Make sure that Parhelia Buffer Conditioner has been added at 1:100 to both buffers * Slide staining: ensure the gray aluminum shelf is correctly positioned in the staining chamber, if this is off the liquid may be applied to the outside of the cover tile rather than into the capillary gap. * Make sure that the sample is positioned correctly within the staining chamber. Coverslip staining: make sure that the coverslip rests evenly on both spacers. ![](/files/tbdFwTWKTYYK1h81WNQO) ### Temperature-controlled protocols * Set up the temperature module on the deck of the Opentrons [following this tutorial](https://www.google.com/url?q=https://support.opentrons.com/s/article/Temperature-Module\&sa=D\&source=editors\&ust=1684536753681232\&usg=AOvVaw0Dot2IzyeznE0dFwQFzMkn). We recommend placing the temperature module in position 7 of the OT-2 Deck * Place thermal sheet on top of the Omni-Stainer * When running the protocols, make sure to select “omni\_stainer\_###\_with\_thermosheath”, where ### is the instrument model (i.e. S12\_slides or C12\_cslps) * During the calibration step, position the pipette tip over the calibration pin * If you are writing your own protocol, make sure to use functions openShutter(...), closeShutter(...) that you can find in the protocol header. Failure to do so may get your pipette damaged and we are not going to be responsible. Besides this email, we have a documentation page that we are actively updating and expanding: [https://parhelia-biosciences.gitbook.io/omni-stainer-user-guide/](https://www.google.com/url?q=https://parhelia-biosciences.gitbook.io/omni-stainer-user-guide/\&sa=D\&source=editors\&ust=1684536753682566\&usg=AOvVaw3kkvsmkMtrOHgv8v3xXwa3) If you identify any missing bits that would be good to cover, please reach out to me or the team at . Yours sincerely, This page was intentionally left blank ©Parhelia Biosciences Corporation, 2022 ![](/files/GraA3M7ZOX9mhinxlPXs)