License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
This protocol describes the antibody conjugation and validation processes for the CODEX® (now PhenoCycler™) system by Akoya Biosciences. For the comprehensive multiplexed imaging workflow currently in use at the Vanderbilt Diabetes Research Center, please see CODEX® Multiplexed Imaging | Modality overview.
Primary antibodies used for conjugation must be free of additives (e.g., BSA, glycerol), so keep this in mind when selecting antibodies to screen. Some manufacturers offer alternate formulations of listed clones or a "carrier-free" format. In our experience, a low concentration of sodium azide is permissible.
Label one tube and and one filter per antibody. Block each filter with 500 µLFilter Blocking Solution, then centrifuge 12.000 x g, 00:02:00. Discard the flow-through.
Measure actual concentration using a NanoDrop™ or comparable system and calculate the volume for 50 µg.
Prepare each antibody in a minimum volume of 100 µL (dilute with 1X PBS if needed). Centrifuge 12.000 x g, 00:08:00 and discard the flow-through.
Reduce antibodies by applying 260 µLReduction Master Mix to each filter/tube unit and incubating for 00:30:00at room temperature. Centrifuge 12.000 x g, 00:08:00 and discard the flow-through.
Prepare oligonucleotide barcodes:
Add 10 µL of nuclease-free water to each barcode vial to dissolve, then add 210 µLConjugation Solution. Pipet up and down gently to dissolve all material.
Add suspended barcode to the top of the corresponding filter unit. Close lid and briefly vortex, then incubate for 00:02:00 at room temperature. Centrifuge 12.000 x g, 00:08:00 and discard the flow-through.
Wash by adding 450 µLPurification Solution to the top of each filter unit. Centrifuge 12.000 x g, 00:08:00 and discard the flow-through.
go to step #7 and repeat x2 for a total of 3 washes/spins.
Label a new collection tube and remove the lid. Add 100 µL of Antibody Storage Solution to each filter unit. Place the new tube face-down on top of the filter unit. Invert the whole apparatus for collection into the new tube (some liquid will come off the filter immediately). Centrifuge 3.000 x g, 00:02:00 and discard the flow-through.
Transfer conjugated antibody (~ 120 µL) to a screw-top storage tube. Label as follows:
Prepare Screening Buffer and then transfer 3 mL aliquots into 6-well plates.
A
B
Total number of samples
4
Total volume (mL)
40
ddH2O (mL)
28
DMSO (mL)
8
10X CODEX buffer (mL)
4
Table 1: Screening Buffer. Copy and paste all cells above into an Excel sheet, then enter value into cell B1. The volumes for each reagent will automatically be returned in cells B3-B5.
Move coverslips through 3 washes (= 3 aliquots Screening Buffer per coverslip).
Prepare Reporter Stock Solution (RSS):
A
B
Total number of samples
4
Total volume (μl)
400
1X CODEX buffer (μl)
379
Assay reagent (μl)
20
Nuclear stain (μl)
1
Table 2: Reporter Stock Solution. Copy and paste all cells above into an Excel sheet, then enter value into cell B1 (up to 12). The volumes for each reagent will automatically be returned in cells B3-B5.
Make up a reporter mix for each coverslip in a light-protected microcentrifuge tube:
100 µL Reporter Stock Solution (RSS)
2.5 µL each CODEX® barcoded reporter (RX); up to 3 RX (one of each fluorophore) per coverslip
Affix a piece of parafilm onto the bench using lab tape. Use a Sharpie to label spaces for each coverslip, then pipet reporter mixes into the corresponding spaces. Using bent-tip tweezers, place each coverslip face-down onto the pooled drop of reporter mix. Cover with a box top or other light-protective structure and incubate for 00:05:00.
go to step #13 to complete 3 washes in screening buffer (can reuse same aliquots from step 13).
Mount coverslips face-down onto microscope slides for imaging. For this step, either 10 µL of 1X CODEX buffer orInvitrogen™ SlowFade™ Gold Antifade MountantFisher ScientificCatalog #S36936 can be used.