💙"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

  

  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. https://youtu.be/hiN0EOW3vHM?t=98

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)

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 guide for more info!

3KB

OMNI_S_12slides_v1.json

Omni-Stainer S12 labware definition

2KB

Parhelia_12trough_reservoir.json

12-trough (AKA 12-well or 12-channel) labware definition

3KB

OMNI_C_12cslps_v1.json

Omni-Stainer C12 labware definition

Remember to add 20-30 ml of distilled water to the Omni-Stainer base.

• 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.

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

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.

• 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 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 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.
• 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

Omni-Stainer™ S12 running Dye Demo (sped up)

Omni-Stainer™ C12 running Dye Demo (sped up)

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.

The Opentrons Deck Calibration Video 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™ which is performed using the embossed "+" symbol on the C12 or S12 Omni-Stainer™ lid.

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:

Omni-Stainer Assembly and Sample Prep reference:

☑️S12 Assembly and Sample Prep

☑️C12 Assembly and Sample Prep