CODEX Staining Protocol For FFPE Tissue

Heat sample coverslip on hot plate at 55°C with tissue facing up for 20-25 minutes until wax thoroughly melts.

Place the sample coverslip in the coverslip staining rack and wait 5 minutes to allow the tissue to cool down.

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

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.

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.

Place staining rack in a beaker containing 40 ml of ddH2O for 2 minutes.

Place the staining rack in a second beaker filled with ddH2O and incubate for 2 minutes.

Place the staining rack in a third beaker filled with 1X PBS and incubate for 2 minutes.

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.

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.

Wash the sample coverslip three times in 1x PBS for 2 minutes.

Immerse the sample coverslip in a well of a 6-well plate containing 5 mls CODEX® Hydration Buffer and incubate for 2 minutes.

Move sample coverslip to a second well containing CODEX® Hydration Buffer and incubate for another 2 minutes.

Move sample coverslip to a well containing CODEX® Staining Buffer and incubate for 20-30 minutes.

Prepare a stock solution of CODEX® Blocking Buffer to be used for the Antibody Cocktail.

A	B
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.

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.

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.

Place sample coverslip in a second well containing CODEX® Staining Buffer for 2 minutes.

Place sample coverslip in a well containing 5 mls of 1.6% PFA in CODEX® Storage Buffer and incubate for 10 minutes.

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.

Transfer coverslip to well of another 6-well plate containing Ice cold methanol and incubate for 5 minutes on ice.

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.

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.

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.

Transfer sample coverslip to a well of a 6-well plate containing 5 mls of CODEX® Storage Buffer.