TITLE: CODEX Staining Protocol For FFPE Tissue
AUTHORS: Kyung J Ahn, Shovik Bandyopadhyay, Anusha           Thadi, Kai  Tan

[DESCRIPTION]
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

[STEPS]
SECTION: Tissue Pre-treatment
1. Heat sample coverslip on hot plate at 55°C with tissue facing up for 20-25 minutes until wax thoroughly melts.
SECTION: Tissue Deparaffinization and Hydration
3. 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
SECTION: Antigen Retrieval
4. 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.
SECTION: Antigen Retrieval
5. Immerse the staining rack in the beaker containing the 1x Citrate buffer and wrap it with aluminum foil to ensure the best sealing possible. 
SECTION: Antigen Retrieval
6. Fill the pressure cooker with water approximately halfway up the height of the beaker. Place the aluminum foil wrapped beaker in the pressure cooker.
SECTION: Antigen Retrieval
7. Set the pressure cooker to the high-pressure protocol and let the tissue incubate for 20 min.
SECTION: Antigen Retrieval
8. 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.
SECTION: Tissue Pre-treatment
2. Place the sample coverslip in the coverslip staining rack and wait 5 minutes to allow the tissue to cool down.
SECTION: Wash Tissue
9. Place staining rack in a beaker containing 40 ml of ddH2O for 2 minutes.
SECTION: Wash Tissue
10. Place the staining rack in a second beaker filled with ddH2O and incubate for 2 minutes.
11. Place the staining rack in a third beaker filled with 1X PBS and incubate for 2 minutes.
SECTION: Photobleach Tissue
12. 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). 
SECTION: Photobleach Tissue
13. Sandwich the 6-well plate between two broad-spectrum LED light sources for 45 minutes at 4°C.
SECTION: Photobleach Tissue
14. 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.
51. 
51. 
51. 
SECTION: Wash Tissue
15. Wash the sample coverslip three times in 1x PBS for 2 minutes.
SECTION: Wash Tissue
16. Immerse the sample coverslip in a well of a 6-well plate containing 5 mls CODEX® Hydration Buffer and incubate for 2 minutes.

SECTION: Wash Tissue
17. Move sample coverslip to a second well containing CODEX® Hydration Buffer and incubate for another 2 minutes.
SECTION: Equilibrate Tissue in Staining Buffer
18. Move sample coverslip to a well containing CODEX® Staining Buffer and incubate for 20-30 minutes.
SECTION: Equilibrate Tissue in Staining Buffer
20. Prepare a stock solution of CODEX Blocking Buffer to be used for the Antibody Cocktail.a.
 
     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   
 
SECTION: Equilibrate Tissue in Staining Buffer
1. a.

   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   

SECTION: Preparation of the Antibody Cocktail Solution
19. 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   
 
19. 
SECTION: Preparation of the Antibody Cocktail Solution
20. 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.
SECTION: Preparation of the Antibody Cocktail Solution
21. Add the appropriate volume of each CODEX Antibody to the Antibody Cocktail Solution.
SECTION: Preparation of the Antibody Cocktail Solution
22. Pipette to mix and briefly spin down the tube.
SECTION: Tissue Staining
23. Remove sample coverslip from the well containing Staining Buffer and place it on the tray of a humidity chamber.
SECTION: Tissue Staining
24. Add 190 μL of the Antibody Cocktail to the top corner of the sample coverslip and ensure that the liquid covers the entire tissue.
SECTION: Tissue Staining
25. Incubate overnight in the humidity chamber at 4°C.
SECTION: Wash Tissue
26. 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.
SECTION: Wash Tissue
27. Place sample coverslip in a second well containing CODEX® Staining Buffer for 2 minutes.
SECTION: Fix Tissue
28. Place sample coverslip in a well containing 5 mls of 1.6% PFA in CODEX® Storage Buffer and incubate for 10 minutes.
SECTION: Wash Tissue
29. Transfer coverslip to a well of a 6-well plate containing 5 mls of 1X PBS and immerse sample coverslip 2-3 times.
SECTION: Wash Tissue
30. Transfer coverslip to a second well of 1X PBS and immerse sample coverslip 2-3 times.
SECTION: Wash Tissue
31. Transfer coverslip to a third well of 1X PBS and immerse sample coverslip 2-3 times.
SECTION: Ice-cold Methanol Incubation
32. Transfer coverslip to well of another 6-well plate containing Ice cold methanol and incubate for 5 minutes on ice.
SECTION: Wash Tissue
33. 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.
SECTION: Wash Tissue
34. Transfer the sample coverslip to the second 1x PBS well and immerse sample coverslip 2-3 times.
SECTION: Wash Tissue
35. Transfer the sample coverslip to the third 1x PBS well and immerse sample coverslip 2-3 times.
SECTION: Fix Tissue
36. Prepare the Final Fixative Solution by diluting the 20 μL of the CODEX® Fixative Reagent in 1 mL of 1x PBS.
SECTION: Fix Tissue
37. Remove sample coverslip from the well and place it on the tray of a humidity chamber.
SECTION: Fix Tissue
38. 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.
SECTION: Fix Tissue
39. Incubate for 20 mins.
SECTION: Wash Tissue
40. 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.
SECTION: Wash Tissue
41. Move the sample coverslip to the second well containing 1x PBS and immerse sample coverslip 2-3 times.
SECTION: Wash Tissue
42. Move the sample coverslip to the third well containing 1x PBS and immerse sample coverslip 2-3 times.
SECTION: Store Tissue
43. Transfer sample coverslip to a well of a 6-well plate containing 5 mls of CODEX® Storage Buffer.