# PhenoCycler® (CODEX) {% 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="../product-guides/blue-dye-demo-automated-protocol" %} [blue-dye-demo-automated-protocol](https://docs.parheliabio.com/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**](https://docs.parheliabio.com/product-guides/s12-assembly-and-sample-prep) section. 2. **For Coverslip-Mounted Samples -** see step 2 in [**C12 Assembly and Sample Prep**](https://docs.parheliabio.com/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="../product-guides/automation-opentrons-ot-2-setup/importing-labware-definitions" %} [importing-labware-definitions](https://docs.parheliabio.com/product-guides/automation-opentrons-ot-2-setup/importing-labware-definitions) {% endcontent-ref %}
--- # Agent Instructions: Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. Perform an HTTP GET request on the current page URL with the `ask` query parameter: ``` GET https://docs.parheliabio.com/protocol-knowledge-base/phenocycler-r-codex.md?ask= ``` The question should be specific, self-contained, and written in natural language. The response will contain a direct answer to the question and relevant excerpts and sources from the documentation. Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.