CODEX® Multiplexed Imaging | Antibody Conjugation and Validation

Establish the efficacy of the primary antibody using an indirect immunofluorescence protocol (e.g., steps 1-20 of Immunofluorescent Staining of Mouse Pancreas).

ⓘ See also: Antibody Screening and Custom-conjugation - Tips and guidelines (Akoya Biosciences).

Gather reagents:

• Oligonucleotide barcodes (Akoya Biosciences)
• 50 μg each primary antibody to be conjugated (vendor of choice)

ⓘ For an overview of the conjugation workflow, see Conjugating CODEX® tags on antibodies of choice (Akoya Biosciences).

Label one tube and and one filter per antibody. Block each filter with 500µL Filter Blocking Solution , then centrifuge 12.000x g. Discard the flow-through.

Measure actual concentration using a NanoDrop™ or comparable system and calculate the volume for 50µg.

Prepare each antibody in a minimum volume of 100µL (dilute with 1X PBS if needed). Centrifuge 12.000x g and discard the flow-through.

Reduce antibodies by applying 260µL Reduction Master Mix to each filter/tube unit and incubating for 0h 30m 0sat room temperature. Centrifuge 12.000x g and discard the flow-through.

Prepare oligonucleotide barcodes:

Add 10µL of nuclease-free water to each barcode vial to dissolve, then add 210µL Conjugation 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 0h 2m 0s at room temperature. Centrifuge 12.000x g and discard the flow-through.

Wash by adding 450µL Purification Solution to the top of each filter unit. Centrifuge 12.000x g and discard the flow-through.

and repeat x2 for a total of 3 washes/spins.

Label a new collection tube and remove the lid. Add 100µL 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 3.000x g and discard the flow-through.

Transfer conjugated antibody (~ 120µL) to a screw-top storage tube. Label as follows:

AB - BX###

Conjugation date

AB lot number

Antibodies should be stored at 4°C.

Follow the staining protocol CODEX® Multiplexed Imaging | Tissue Staining and Reporter Plate Preparation through step 20.

Prepare Screening Buffer and then transfer 3mL aliquots into 6-well plates.

A	B
Total number of samples	4
Total volume (mL)	=B1*10
ddH2O (mL)	=B2-(B4+B5)
DMSO (mL)	=B2/5
10X CODEX buffer (mL)	=B2/10

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)	=B1*100
1X CODEX buffer (μl)	=B2-(B4+B5)
Assay reagent (μl)	=B2/20
Nuclear stain (μl)	=IF(B1<7,1,2)

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:

100µL Reporter Stock Solution (RSS)

2.5µL 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 0h 5m 0s.

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 10µL of 1X CODEX buffer or can be used.