TITLE: CODEX® Multiplexed Imaging | Antibody Conjugation and Validation
AUTHORS: Diane  Saunders, Conrad Reihsmann, Marcela Brissova, Alvin C. Powers

[DESCRIPTION]
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.

[STEPS]
SECTION: Custom Antibody Conjugations
2. 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).
SECTION: Custom Antibody Conjugations
4. 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, 8 min and discard the flow-through.
SECTION: Custom Antibody Conjugations
3. Label one tube and and one filter per antibody. Block each filter with 500 µL Filter Blocking Solution, then centrifuge 12.000 x g, 2 min. Discard the flow-through.
SECTION: Custom Antibody Conjugations
5. Reduce antibodies by applying 260 µL Reduction Master Mix to each filter/tube unit and incubating for 30 minat room temperature. Centrifuge 12.000 x g, 8 min and discard the flow-through.
SECTION: Custom Antibody Conjugations
6. Prepare oligonucleotide barcodes:

Add 10 µL of nuclease-free water to each barcode vial to dissolve, then add 210 µL 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 2 min at room temperature. Centrifuge 12.000 x g, 8 min and discard the flow-through.
SECTION: Custom Antibody Conjugations
7. Wash by adding 450 µL Purification Solution to the top of each filter unit. Centrifuge 12.000 x g, 8 min and discard the flow-through.
SECTION: Custom Antibody Conjugations
8.   and repeat x2 for a total of 3 washes/spins.

SECTION: Custom Antibody Conjugations
9. 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, 2 min and discard the flow-through.

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

AB - BX###
Conjugation date
AB lot number

Antibodies should be stored at 4 °C.

SECTION: Conjugated Antibody Screening
11. Follow the staining protocol CODEX® Multiplexed Imaging | Tissue Staining and Reporter Plate Preparation through step 20.
SECTION: Conjugated Antibody Screening
12. Prepare Screening Buffer and then transfer 3 mL aliquots into 6-well plates.

 

SECTION: Conjugated Antibody Screening
13. Move coverslips through 3 washes (= 3 aliquots Screening Buffer per coverslip).
SECTION: Conjugated Antibody Screening
14. Prepare Reporter Stock Solution (RSS):


 

SECTION: Conjugated Antibody Screening
15. 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
SECTION: Conjugated Antibody Screening
16. 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 5 min.
SECTION: Conjugated Antibody Screening
17.   to complete 3 washes in screening buffer (can reuse same aliquots from step 13).
SECTION: Conjugated Antibody Screening
18. Mount coverslips face-down onto microscope slides for imaging. For this step, either 10 µL of 1X CODEX buffer or  can be used.
SECTION: Primary Antibody Screening
1. 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).