Enable Medicine offers lab services for the generation of highplex fluorescence IHC images via CODEX. This is an excellent option for those who require images of their samples but lack the necessary equipment or resources. This page offers a brief summary of the CODEX workflow utilized by the Enable Lab. Antibody conjugation, tissue staining, and image acquisition are performed in the Enable Lab according to Akoya's user manuals (CODEX User Manual Rev C for PhenoCycler-Open, and PhenoCycler-Fusion User Guide Rev F for PhenoCycler-Fusion) (https://www.akoyabio.com).
Sample preparation.
PhenoCycler-Open: To improve adherence of tissue sections prior to mounting, glass coverslips for samples are coated with 0.1% poly-L-lysine solution. The prepared coverslips are washed and stored as per the guidelines in the CODEX User Manual. For preparing tissue sections on coverslips, please see our Sample Preparation Guidelines.
PhenoCycler-Fusion: Samples for mounting on slides are cut onto standard 1”x3” positively charged slides.
Antibody conjugation with barcodes.
For all custom biomarkers, the antibodies are conjugated in the Enable Lab using the CODEX® Conjugation Kit, following the conjugation protocol provided in the User Manual. Briefly, (1) the antibody is partially reduced to expose thiol ends of the antibody heavy chains; (2) the reduced antibody is conjugated with a CODEX barcode; (3) the conjugated antibody is purified; and (4) Antibody Storage Solution is added for antibody stabilization for long term storage. This diagram taken directly from the manual provides an overview of the conjugation process:
When we purchase antibodies for in-house conjugations, we select purified antibodies that are free of carrier proteins (i.e. BSA-, Gelatin-, Glycerol-free), as these proteins can interfere with the conjugation process.
To verify the successful conjugation of barcodes to antibodies, we run a gel electrophoresis with both the pre-conjugated and post-conjugated antibodies. The image below, taken from the Akoya user manual, shows what the gel looks like with successful conjugation:
Finally, the conjugated antibodies are validated in a QC stain. For more information, please refer to the following page
Staining
Sample coverslips/slides are stained following the protocols provided by Akoya. Briefly, samples are heated on a hot plate to bake the tissue (55°C for 20-25 minutes for coverslips, 60°C overnight for slides). The samples undergo deparaffinization and rehydration in a series of ethanol washes, before being subject to antigen retrieval in 1X Tris-EDTA buffer (pH = 9.0). The samples are then blocked in staining buffer and incubated with antibody cocktail for 3 hours in room temperature. After incubation, the samples are washed and fixed following the user manual.
Image acquisition and processing
PhenoCycler-Open: Sample coverslips are placed on a microscope stage, and images are captured using a Keyence microscope that is configured to the PhenoCycler Instrument with a 20X objective. The raw .tif files are transferred from the Codex Instrument Manager software and processed using Enable’s internal AWS processing pipeline, including pre-processing for drift correction, background subtraction, and tile stitching to finally generate hyperstacked images that are viewable in the Visualizer.