May 10, 2022

Public workspaceCODEX® Multiplexed Imaging | Antibody Conjugation and Validation

DOI
dx.doi.org/10.17504/protocols.io.kxygxzwmzv8j/v1
  • 1Vanderbilt University, Vanderbilt University Medical Center;
  • 2Vanderbilt University Medical Center
Islet and Pancreas Analysis Core
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DOI: dx.doi.org/10.17504/protocols.io.kxygxzwmzv8j/v1
Protocol Citation: Diane Saunders, Conrad Reihsmann, Marcela Brissova, Alvin C. Powers 2022. CODEX® Multiplexed Imaging | Antibody Conjugation and Validation. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxzwmzv8j/v1
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
Protocol status: Working
We use this protocol and it’s working
Created: March 25, 2022
Last Modified: May 10, 2022
Protocol Integer ID: 59932
Disclaimer
This protocol is adapted from the CODEX User Manual, revision C (Akoya Biosciences, Dec. 2020).
Abstract
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.
Materials
For Conjugation:

  • Reagent50kDa MWCO filterEmd MilliporeCatalog #UFC505096
  • ReagentCODEX® Conjugation KitAkoya BiosciencesCatalog #7000009
Contains: Filter Blocking Solution, Reduction Master Mix, Conjugation Solution, Purification Solution, Antibody Storage Solution

  • CODEX® oligonucleotide barcodes
  • Primary antibodies (50 μg each)


For Validation:

  • Reagent10X CODEX BufferAkoya BiosciencesCatalog #7000001
  • ReagentAssay ReagentAkoya BiosciencesCatalog #7000002
  • ReagentNuclear StainAkoya BiosciencesCatalog #7000003
  • ReagentDimethyl sulfoxide ≥99.9%Sigma AldrichCatalog #472301-6X1L
  • ReagentInvitrogen™ SlowFade™ Gold Antifade MountantFisher ScientificCatalog #S36936
  • CODEX® barcoded reporters
  • See CODEX® Multiplexed Imaging | Tissue Staining and Reporter Plate Preparation for complete list


Equipment:

  • Benchtop microcentrifuge
  • Nanodrop

Protocol materials
ReagentInvitrogen™ SlowFade™ Gold Antifade MountantFisher ScientificCatalog #S36936
Materials, Step 18
Reagent50kDa MWCO filterMerck Millipore (EMD Millipore)Catalog #UFC505096
Materials, Step 2
ReagentCODEX® Conjugation KitAkoya BiosciencesCatalog #7000009
Materials, Step 2
Reagent10X CODEX BufferAkoya BiosciencesCatalog #7000001
Materials
ReagentAssay ReagentAkoya BiosciencesCatalog #7000002
Materials
ReagentNuclear StainAkoya BiosciencesCatalog #7000003
Materials
ReagentDimethyl sulfoxide ≥99.9%Merck MilliporeSigma (Sigma-Aldrich)Catalog #472301-6X1L
Materials
Primary Antibody Screening
Primary Antibody Screening
Establish the efficacy of the primary antibody using an indirect immunofluorescence protocol (e.g., steps 1-20 of Immunofluorescent Staining of Mouse Pancreas).
Safety information
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.

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

Custom Antibody Conjugations
Custom Antibody Conjugations
Gather reagents:
  • ReagentCODEX® Conjugation KitAkoya BiosciencesCatalog #7000009
  • Reagent50kDa MWCO filterEmd MilliporeCatalog #UFC505096
  • 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 Amount500 µL Filter Blocking Solution, then centrifuge Centrifigation12.000 x g, 00:02:00 . Discard the flow-through.
Measure actual concentration using a NanoDrop™ or comparable system and calculate the volume for Amount50 µg .

Prepare each antibody in a minimum volume of Amount100 µL (dilute with 1X PBS if needed). Centrifuge Centrifigation12.000 x g, 00:08:00 and discard the flow-through.
Reduce antibodies by applying Amount260 µL Reduction Master Mix to each filter/tube unit and incubating for Duration00:30:00 at room temperature. Centrifuge Centrifigation12.000 x g, 00:08:00 and discard the flow-through.
Prepare oligonucleotide barcodes:

Add Amount10 µL of nuclease-free water to each barcode vial to dissolve, then add Amount210 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 Duration00:02:00 at room temperature. Centrifuge Centrifigation12.000 x g, 00:08:00 and discard the flow-through.
Wash by adding Amount450 µL Purification Solution to the top of each filter unit. Centrifuge Centrifigation12.000 x g, 00:08:00 and discard the flow-through.
Go togo to step #7 and repeat x2 for a total of 3 washes/spins.

Label a new collection tube and remove the lid. Add Amount100 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 Centrifigation3.000 x g, 00:02:00 and discard the flow-through.

Transfer conjugated antibody (~ Amount120 µL ) to a screw-top storage tube. Label as follows:

AB - BX###
Conjugation date
AB lot number

Antibodies should be stored at Temperature4 °C .

Conjugated Antibody Screening
Conjugated Antibody Screening
Prepare Screening Buffer and then transfer Amount3 mL aliquots into 6-well plates.

AB
Total number of samples4
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):


AB
Total number of samples4
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:

Amount100 µL Reporter Stock Solution (RSS)
Amount2.5 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 Duration00:05:00 .
Go togo 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 Amount10 µL of 1X CODEX buffer orReagentInvitrogen™ SlowFade™ Gold Antifade MountantFisher ScientificCatalog #S36936 can be used.