Apr 29, 2022
CODEX Staining Protocol For FFPE Tissue
- Kyung J Ahn1,
- Shovik Bandyopadhyay2,
- Anusha Thadi1,
- Kai Tan1
- 1Children's Hospital of Philadelphia;
- 2University of Pennsylvania
- Human BioMolecular Atlas Program (HuBMAP) Method Development Community
- CHOP TMC CODEX on FFPE Tissue
Protocol Citation: Kyung J Ahn, Shovik Bandyopadhyay, Anusha Thadi, Kai Tan 2022. CODEX Staining Protocol For FFPE Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzyjn4lx1/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 11, 2022
Last Modified: April 29, 2022
Protocol Integer ID: 59374
Keywords: HuBMAP, CHOP TMC, CODEX, HTAN
Funders Acknowledgements:
National Institutes of Health
Grant ID: U2CCA233285
National Institutes of Health
Grant ID: U54HL156090
Abstract
This protocol describes the method for antibody staining of FFPE tissues on coverslips using CODEX Barcoded Antibodies. The Akoya CODEX user manual was modified to include photobleaching steps. Included are the stepwise protocols for pre-staining, deparaffinization, antigen retrieval, photobleaching, antibody staining and post-fixation.
Guidelines
- Use poly-l-Lysine coated coverslips.
- Keep tissue sections from drying during transfer steps
Materials
CODEX® Staining Kit (Akoya PN: 7000008)
Tissue Pre-treatment
Tissue Pre-treatment
25m
25m
Heat sample coverslip on hot plate at 55°C with tissue facing up for 20-25 minutes until wax thoroughly melts.
20m
Place the sample coverslip in the coverslip staining rack and wait 5 minutes to allow the tissue to cool down.
5m
Tissue Deparaffinization and Hydration
Tissue Deparaffinization and Hydration
50m
50m
Immerse the staining rack in the container containing the following reagents for 5 minutes each:
a.Xylene
b.Xylene
c.100% Ethanol/Reagent Alcohol
d.100% Ethanol/Reagent Alcohol
e.90% Ethanol/Reagent Alcohol
f.70% Ethanol/Reagent Alcohol
g.50% Ethanol/Reagent Alcohol
h.30% Ethanol/Reagent Alcohol
i.ddH2O
j.ddH2O
50m
Antigen Retrieval
Antigen Retrieval
45m
45m
In a 50 ml Pyrex beaker, prepare 40 mls of a 1x citrate buffer (0.01 M). Dilute 100x citrate buffer pH6.0 to 1X citrate buffer in ddH2O.
Immerse the staining rack in the beaker containing the 1x Citrate buffer and wrap it with aluminum foil to ensure the best sealing possible.
Fill the pressure cooker with water approximately halfway up the height of the beaker. Place the aluminum foil wrapped beaker in the pressure cooker.
Set the pressure cooker to the high-pressure protocol and let the tissue incubate for 20 min.
30m
After incubation in the pressure cooker, release the pressure and carefully remove the rack from the pressure cooker and allow to cool on the bench for 15 minutes until no longer hot to the touch.
15m
Wash Tissue
Wash Tissue
6m
6m
Place staining rack in a beaker containing 40 ml of ddH2O for 2 minutes.
2m
Place the staining rack in a second beaker filled with ddH2O and incubate for 2 minutes.
2m
Place the staining rack in a third beaker filled with 1X PBS and incubate for 2 minutes.
2m
Photobleach Tissue
Photobleach Tissue
1h 30m
1h 30m
Submerge the sample coverslip in a well of a 6-well plate containing 5 mls of 4.5% (w/v) H2O2 and 20mM NaOH in PBS (bleaching solution).
Sandwich the 6-well plate between two broad-spectrum LED light sources for 45 minutes at 4°C.
45m
After 45 minutes move the sample coverslip(s) into a new well with fresh bleaching solution and photobleach for another 45 minutes at 4°C.
45m
Wash Tissue
Wash Tissue
10m
10m
Wash the sample coverslip three times in 1x PBS for 2 minutes.
6m
Immerse the sample coverslip in a well of a 6-well plate containing 5 mls CODEX® Hydration Buffer and incubate for 2 minutes.
2m
Move sample coverslip to a second well containing CODEX® Hydration Buffer and incubate for another 2 minutes.
2m
Equilibrate Tissue in Staining Buffer
Equilibrate Tissue in Staining Buffer
20m
20m
Move sample coverslip to a well containing CODEX® Staining Buffer and incubate for 20-30 minutes.
20m
Preparation of the Antibody Cocktail Solution
Preparation of the Antibody Cocktail Solution
Prepare a stock solution of CODEX® Blocking Buffer to be used for the Antibody Cocktail.
| CODEX® Blocking Buffer | 1 Sample | |
| Staining Buffer [µL] | 181 | |
| N Blocker [µL] | 4.75 | |
| G Blocker [µL] | 4.75 | |
| J Blocker [µL] | 4.75 | |
| S Blocker [µL] | 4.75 | |
| Total [µL] | 200 |
Add CODEX® Blocking Buffer to a tube designated for Antibody Cocktail Staining Solution. The volume of CODEX® Blocking Buffer to be prepared for each sample coverslip can vary depending on the titer and corresponding volume of each antibody. Adjust volume of CODEX® Blocking Buffer so that the final volume of the Antibody Cocktail Staining Solution is a total of 200 μL per tissue.
Add the appropriate volume of each CODEX Antibody to the Antibody Cocktail Solution.
Pipette to mix and briefly spin down the tube.
Tissue Staining
Tissue Staining
16h
16h
Remove sample coverslip from the well containing Staining Buffer and place it on the tray of a humidity chamber.
Add 190 μL of the Antibody Cocktail to the top corner of the sample coverslip and ensure that the liquid covers the entire tissue.
Incubate overnight in the humidity chamber at 4°C.
16h
Wash Tissue
Wash Tissue
4m
4m
After overnight antibody incubation, immerse the sample coverslip in a well of a 6-well plate containing 5 mls CODEX® Staining Buffer for 2 minutes.
2m
Place sample coverslip in a second well containing CODEX® Staining Buffer for 2 minutes.
2m
Fix Tissue
Fix Tissue
10m
10m
Place sample coverslip in a well containing 5 mls of 1.6% PFA in CODEX® Storage Buffer and incubate for 10 minutes.
10m
Wash Tissue
Wash Tissue
1m
1m
Transfer coverslip to a well of a 6-well plate containing 5 mls of 1X PBS and immerse sample coverslip 2-3 times.
Transfer coverslip to a second well of 1X PBS and immerse sample coverslip 2-3 times.
Transfer coverslip to a third well of 1X PBS and immerse sample coverslip 2-3 times.
Ice-cold Methanol Incubation
Ice-cold Methanol Incubation
5m
5m
Transfer coverslip to well of another 6-well plate containing Ice cold methanol and incubate for 5 minutes on ice.
5m
Wash Tissue
Wash Tissue
1m
1m
Place the 6-well plate containing the previously used 1x PBS next to the ice bucket and the methanol tray containing the sample coverslip. Quickly transfer the sample coverslip from methanol to the first corresponding 1x PBS well and immerse sample coverslip 2-3 times.
Transfer the sample coverslip to the second 1x PBS well and immerse sample coverslip 2-3 times.
Transfer the sample coverslip to the third 1x PBS well and immerse sample coverslip 2-3 times.
Fix Tissue
Fix Tissue
20m
20m
Prepare the Final Fixative Solution by diluting the 20 μL of the CODEX® Fixative Reagent in 1 mL of 1x PBS.
Remove sample coverslip from the well and place it on the tray of a humidity chamber.
Add 200 μL of Final Fixative Solution to the top corner of the sample coverslip and ensure that the entire tissue is covered in fixative solution.
Incubate for 20 mins.
20m
Wash Tissue
Wash Tissue
1m
1m
Remove the sample coverslip from the humidity chamber and place in the first well containing previously used 1x PBS. Lift and immerse the sample coverslip 2-3 times.
Move the sample coverslip to the second well containing 1x PBS and immerse sample coverslip 2-3 times.
Move the sample coverslip to the third well containing 1x PBS and immerse sample coverslip 2-3 times.
Store Tissue
Store Tissue
Transfer sample coverslip to a well of a 6-well plate containing 5 mls of CODEX® Storage Buffer.