May 10, 2022

Public workspaceCODEX® Multiplexed Imaging | Tissue Staining and Reporter Plate Preparation 

DOI
dx.doi.org/10.17504/protocols.io.n92ldzro9v5b/v1
  • 1Vanderbilt University, Vanderbilt University Medical Center;
  • 2Vanderbilt University Medical Center
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DOI: dx.doi.org/10.17504/protocols.io.n92ldzro9v5b/v1
Protocol Citation: Diane Saunders, Conrad Reihsmann, Marcela Brissova, Alvin C. Powers 2022. CODEX® Multiplexed Imaging | Tissue Staining and Reporter Plate Preparation . protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldzro9v5b/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: April 07, 2022
Last Modified: May 10, 2022
Protocol Integer ID: 60459
Disclaimer
This protocol is adapted from the CODEX User Manual, revision C (Akoya Biosciences, Dec. 2020). For a quick reference and overview, see FF Tissue Staining and Reporter Plate Preparation. Although the protocol is designed for flash frozen (unfixed) tissue, we have validated that it also works for lightly PFA-fixed tissue.
Abstract
This protocol describes the staining and pre-imaging preparation currently in use by the Vanderbilt Diabetes Research Center Islet & Pancreas Analysis (IPA) Core and Powers/Brissova Research Group prior to performing multiplexed imaging of the human pancreas using the CO-Detection by indEXing (CODEX®) platform (now PhenoCycler™; Akoya Biosciences). See also CODEX® Multiplexed Imaging | Modality overview.
Materials
CODEX® Supplies:

  • Reagent10X CODEX BufferAkoya BiosciencesCatalog #7000001
  • ReagentAssay ReagentAkoya BiosciencesCatalog #7000002
  • ReagentNuclear StainAkoya BiosciencesCatalog #7000003
  • Reagent96 well plateAkoya BiosciencesCatalog #7000006
  • Reagent96 well Plate Foil SealsAkoya BiosciencesCatalog #7000007
  • ReagentCODEX Staining kit Akoya BiosciencesCatalog #7000008
Contains: Hydration Buffer, Staining Buffer, Storage Buffer, N/J/G/S Blockers, Fixative reagent

  • Primary antibodies with CODEX® oligonucleotide barcodes
  • CODEX® barcoded reporters


Additional Reagents and Consumables:

  • Reagent1X Dulbecco’s Phosphate Buffered Saline (DPBS) Thermo Fisher ScientificCatalog #14190094
  • Reagent16% ParaformaldehydeFisher ScientificCatalog #15710
  • ReagentBent-tip tweezersFine Science ToolsCatalog #11251-33
  • ReagentAcetoneSigma AldrichCatalog #650501-1L
  • ReagentMethanolSigma AldrichCatalog #34860-1L-R
  • ReagentDrierite™ indicating absorbentsFisher ScientificCatalog #23-116582
  • ReagentAmber 1.5-mL tubesVWR ScientificCatalog #89000-030
  • Nuclease free water
  • 6 well plates - non-tissue culture treated


Additional Supplies:

  • 15-mL glass beakers
Protocol materials
Reagent10X CODEX BufferAkoya BiosciencesCatalog #7000001
Materials
ReagentAmber 1.5-mL tubesVWR InternationalCatalog #89000-030
Materials
ReagentCODEX Staining kit Akoya BiosciencesCatalog #7000008
Materials
Reagent1X Dulbecco’s Phosphate Buffered Saline (DPBS) Thermo Fisher ScientificCatalog #14190094
Materials
ReagentAcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #650501-1L
Materials
ReagentBent-tip tweezersFine Science ToolsCatalog #11251-33
Materials
ReagentDrierite™ indicating absorbentsFisher ScientificCatalog #23-116582
Materials, Step 2
Reagent16% ParaformaldehydeFisher ScientificCatalog #15710
Materials
Reagent96 well plateAkoya BiosciencesCatalog #7000006
Materials, Step 23
Reagent96 well Plate Foil SealsAkoya BiosciencesCatalog #7000007
Materials, Step 23
ReagentAssay ReagentAkoya BiosciencesCatalog #7000002
Materials, Step 23
ReagentMethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #34860-1L-R
Materials
ReagentNuclear StainAkoya BiosciencesCatalog #7000003
Materials, Step 23
Tissue Preparation
Tissue Preparation
Prepare pre-staining reagents (per individual coverslip):

  • 15-mL beaker with Amount10 mL acetone
Note
After acetone fixation, all incubations and washes in this portion of the protocol are done in 6-well plates using approximately Amount3 mL of reagent per well (one well per coverslip). If using an alternative container, this volume must be sufficient to submerge the coverslip.


  • 2 wells Hydration Buffer
  • 1 well Pre-staining Fixation Solution (1.6% PFA in Hydration Buffer)
  • 1 well Staining Buffer
Remove coverslip from freezer and place face up in container with layer ofReagentDrierite™ indicating absorbentsVWR InternationalCatalog #23-116582 for Duration00:02:00 to dry.
Place coverslip into an individual 10-mL beaker containing acetone, making sure entire tissue area is submerged. Incubate for Duration00:10:00 .
Carefully remove coverslip from beaker and place back into Drierite container for another Duration00:02:00 .
Transfer coverslip to 6-well plate and perform two washes in 3 mL Hydration Buffer, incubating for Duration00:02:00 each time. If staining more than one sample at a time, make sure coverslip order/orientation in 6-well plates is kept consistent so you don't lose track of samples.
From second well of Hydration Buffer, transfer coverslip to well containing 3 mL Pre-Staining Fixation Solution (Concentration1.6 % (v/v) PFA in Hydration Buffer) and incubate for Duration00:10:00 .
Wash coverslip twice in 3 mL Hydration Buffer to remove fixative. No incubation is required, and aliquots from step 5 can be reused.
Incubate coverslip in 3 mL Staining Buffer for Duration00:20:00 . Place 6-well plate onto a fixed-angle rocker for the incubation period.
Primary Antibody Stain
Primary Antibody Stain
Prepare CODEX Blocking Buffer for primary antibody cocktail:

  • Staining Buffer - Concentration91 % (v/v)
  • N/G/J/S blockers - Concentration2.4 % (v/v) each

AB
Total number of samples1
Total volume (μl)210
Staining Buffer (μl)190
N blocker (μl)5
G blocker (μl)5
J blocker (μl)5
S blocker (μl)5
Table 1: Blocking 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 B2-B7.

Calculate required amounts for each primary antibody (based on optimized dilution) as well as the total antibody volume. Remove the total volume from the Blocking Buffer, then add primary antibodies. Vortex gently.

ⓘ Preconjugated antibody dilutions: PhenoCycler Antibody Dilutions
Place coverslip face up in humidity chamber (can use an empty pipet tip box with damp paper towel in the bottom; coverslips rest on the removable insert). Carefully pipette Amount200 µL of antibody cocktail solution on top, making sure solution pools to cover the entire tissue area.


Figure 1. Schematic of a humidity chamber made from an empty pipet tip box with coverslips arranged on top of the insert (left). Right panel shows how solution should be pipetted to pool and cover the tissue for incubation.
Incubate at TemperatureRoom temperature for Duration03:00:00 .
Post-Stain Procedure
Post-Stain Procedure
Prepare post-staining reagents (per individual coverslip):
Note
All incubations and washes in this portion of the protocol, with the exception of Final Fixative, are done in 6-well plates using approximately Amount3 mL of reagent per well (one well per coverslip). If using an alternative container, this volume must be sufficient to submerge the coverslip.

  • 1 well Post-staining Fixation Solution (1.6% PFA in Staining Buffer)
  • 2 wells Staining Buffer
  • 1 well methanol TemperatureOn ice
  • 3 wells 1X DPBS
Transfer coverslip from humidity chamber to 6-well plate and perform two washes in 3 mL Staining Buffer, incubating for Duration00:02:00 each time.
From second well of Staining Buffer, transfer coverslip to well containing 3 mL Post-Staining Fixation Solution (Concentration1.6 % (v/v) PFA in Staining Buffer) and incubate for Duration00:10:00 .
Wash coverslip three times in 3 mL DPBS to remove fixative. No incubation is required.
Incubate coverslip in 3 mL cold methanol TemperatureOn ice for Duration00:05:00 .
Wash coverslip three times in 3 mL DPBS to remove methanol. No incubation is required, and aliquots from step 16 can be reused.
Prepare Final Fixative (Concentration2 % (v/v) Fixative Reagent in DPBS).

⚠ Thaw Fixative Reagent right before use and discard any unused volume. One tube provides adequate volume to fix up to 5 samples.
Place coverslip face-up in humidity chamber and carefully pipette Amount200 µL of Final Fixative onto coverslip, making sure it covers the entire tissue area. Incubate at TemperatureRoom temperature for Duration00:20:00 .

20m
Wash coverslip three times in 3 mL DPBS to remove fixative. No incubation is required, and aliquots from steps 16/18 can be reused.
Transfer coverslip into 3 mL of Storage Buffer and label 6-well plate. Add a piece of parafilm before placing lid to ensure solution does not evaporate. Store at Temperature4 °C for up to 2 weeks. Label the top of the plate with Sharpie or tape to keep track of sample(s).
Preparation of Reporter Plate
Preparation of Reporter Plate
Gather supplies and reagents:

  • ReagentNuclear StainVWR InternationalCatalog #7000003
  • ReagentAssay ReagentVWR InternationalCatalog #7000002
  • Reagent96 well plateVWR InternationalCatalog #7000006
  • Reagent96 well Plate Foil SealsVWR InternationalCatalog #7000007


Prepare Reporter Stock Solution:

  • Nuclease-free water - Concentration81.33 % (v/v)
  • 10X CODEX Buffer - Concentration10.00 % (v/v)
  • Assay Reagent - Concentration8.33 % (v/v)
  • CODEX Nuclear Stain - Concentration0.33 % (v/v)

AB
Total number of samples3
Reporter cycles per sample10
Total volume (mL)9
Nuclease-free water (mL)7.32
10X CODEX buffer (μl)900
Assay Reagent (μl)750
CODEX Nuclear Stain (μl)30
Table 2: Reporter Stock Solution. Copy and paste all cells above into an Excel sheet, then enter values into cells B1-B2. The volumes for each reagent will automatically be returned in cells B3-B7.

⚠ Ensure you prepare adequate Reporter Stock Solution to account for Blank cycles (no reporters); formulas in Table 2 reflect this.
Label one 1.5 mL light-blocking microcentrifuge tube for each reporter cycle and aliquot Reporter Stock Solution (Amount250 µL per sample). You should have Reporter Stock Solution remaining (for Blank cycles) after aliquoting.
Prepare reporter mixes by adding appropriate reporters to microcentrifuge tubes (Amount5 µL per sample for each reporter):

AB
Total number of samples3
Total volume Reporter Stock Solution (μl)750
Reporter for channel 1 (μl)15
Reporter for channel 2 (μl)15
Reporter for channel 3 (μl)15
Table 3: Reporter mixes (per cycle). 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 B2-B5.

⚠ Make sure to vortex reporter tubes briefly before adding to reporter mix.
Mix contents of tubes by gently pipetting up and down with pipette or by brief vortex. Spin tubes using benchtop microcentrifuge to collect solution at bottom of tubes.
Add Amount250 µL of reporter mix to each corresponding well in a light-protected 96-well plate, making sure to leave blank wells before and after reporter mixes. Fill these wells (corresponding to Blank cycles) with Amount250 µL Reporter Stock Solution (no reporters in solution).


Figure 2. Schematic of 96-well Reporter Plate. In this example a total of 24 primary antibodies will be visualized in eight imaging cycles, with additional cycles before (#1) and after (#10) serving as "Blank" cycles with DAPI only. After reporter mixes are made up in tubes, each mix (labeled by its cycle number, #2-9) is transferred to its respective well (B2-B9). Reporter Stock Solution is added to wells B1 and B10 for Blank cycles.
Seal plate with foil plate seal and store at Temperature4 °C until imaging.