TITLE: Phenocycler-Fusion 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 slides using CODEX barcoded antibodies.  The Akoya Phenocycler-Fusion 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 positively charged slides.
Keep tissue sections from drying during transfer steps

[STEPS]
SECTION: Tissue Pre-treatment
1. Bake sample slide/s in an incubator at 65°C for 1-3 hours until paraffin thoroughly melts.
SECTION: Tissue Deparaffinization and Hydration
3. Immerse sample slide/s in a coplin jar containing the following reagents for 5 minutes each: 

a.Histochoice 
b.Histochoice
c.100% Ethanol
d.100% Ethanol
e.90% Ethanol
f.70% Ethanol
g.50% Ethanol
h.30% Ethanol
i.ddH2O 
j.ddH2O
SECTION: Antigen Retrieval
4. In a coplin jar, prepare 50 mls of a 1x citrate buffer. Dilute 10x citrate buffer pH6.0 to 1X citrate buffer in ddH2O. 
SECTION: Antigen Retrieval
5. Immerse sample slide/s in the coplin jar containing the 1x Citrate buffer and cover the coplin jar with aluminum foil. 
SECTION: Antigen Retrieval
6. Fill the pressure cooker with 1200 mls of ddH2O. Place the aluminum foil covered coplin jar containing sample slide/s in the pressure cooker.
SECTION: Antigen Retrieval
7. Set the pressure cooker to the high-pressure protocol and let the tissue incubate for 20 minutes.
SECTION: Antigen Retrieval
8. After incubation in the pressure cooker, release the pressure and carefully remove the coplin jar from the pressure cooker and allow to cool at room temperature for 30 minutes.
SECTION: Tissue Pre-treatment
2. Cool sample slide/s at room temperature for 5 minutes to allow the sample slide/s to cool to room temperature.
SECTION: Wash Tissue
9. Place sample slide/s in a coplin jar containing 50 mls of ddH2O for 2 minutes.  All volumes for coplin jar incubations will be 50 mls henceforth in this protocol.
SECTION: Wash Tissue
10. Place sample slide/s in a second coplin jar filled with ddH2O and incubate for 2 minutes.
11. Place sample slide/s in a third coplin jar filled with 1X PBS and incubate for 2 minutes.
SECTION: Photobleach Tissue
12. Submerge the sample slide/s in a 150 mm petri dish containing 50 mls of 4.5% (w/v) H2O2 and 20mM NaOH in PBS (bleaching solution). 
SECTION: Photobleach Tissue
13. Sandwich the petri dish 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 petri dish with fresh bleaching solution and photobleach for another 45 minutes at 4°C.
SECTION: Wash Tissue
15. Wash the sample slide/s three times for 2 minutes each in coplin jars containing 1X PBS.
SECTION: Wash Tissue
16. Immerse the sample slide/s in a coplin jar containing 50 mls of CODEX® Hydration Buffer and incubate for 2 minutes.

SECTION: Wash Tissue
17. Move sample slide/s to a second coplin jar containing CODEX® Hydration Buffer and incubate for another 2 minutes.
SECTION: Equilibrate Tissue in Staining Buffer
18. Move sample sample slide/s to a coplin jar containing 50 mls of CODEX® Staining Buffer and incubate for 20-30 minutes.
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   
 
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 slide/s 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 slide/s from the coplin jar 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 each of the sample slide(s) 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 slide/s in a coplin jar containing 50 mls of CODEX® Staining Buffer for 2 minutes.
SECTION: Wash Tissue
27. Place sample slide/s in a second coplin jar containing CODEX® Staining Buffer for 2 minutes.
SECTION: Fix Tissue
28. Place sample slide/s in a coplin jar containing 50 mls of 1.6% PFA in CODEX® Storage Buffer and incubate for 10 minutes.
SECTION: Wash Tissue
29. Transfer sample slide/s to a coplin jar containing 50 mls of 1X PBS.  Lift and immerse the sample slide/s 2-3 times.
SECTION: Wash Tissue
30. Transfer sample slide/s to a second coplin jar of 1X PBS and immerse sample slide/s 2-3 times.
SECTION: Wash Tissue
31. Transfer sample slide/s to a third coplin jar of 1X PBS and immerse sample slide/s 2-3 times.
SECTION: Ice-cold Methanol Incubation
32. Transfer sample slide/s to a coplin jar containing Ice cold methanol and incubate for 5 minutes on ice.
SECTION: Wash Tissue
33. Place a coplin jar of 1x PBS next to the methanol coplin jar containing the sample slide/s.  Quickly transfer the sample slide/s from methanol to the coplin jar of 1x PBS and immerse sample slide/s 2-3 times.
SECTION: Wash Tissue
34. Transfer the sample slide/s to a second coplin jar of 1x PBS and immerse sample slide/s 2-3 times.
SECTION: Wash Tissue
35. Transfer the sample coverslip to a third coplin jar of 1x PBS and immerse sample slide/s 2-3 times.
SECTION: Fix Tissue
36. Prepare the Final Fixative Solution by diluting 20 μl of the CODEX® Fixative Reagent in 1 ml of 1x PBS (Final Fixative Solution).
SECTION: Fix Tissue
37. Remove sample slide/s from the coplin jar and place it on the tray of a humidity chamber.
SECTION: Fix Tissue
38. Add 200 μL of Final Fixative Solution to each of the sample slide/s 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 slide/s from the humidity chamber and place in a coplin jar containing 1x PBS. Lift and immerse the sample slide/s 2-3 times.
SECTION: Wash Tissue
41. Move the sample slide/s to a second coplin jar containing 1x PBS and immerse sample slide/s 2-3 times.
SECTION: Wash Tissue
42. Move the sample slide/s to a third coplin jar containing 1x PBS and immerse sample slide/s 2-3 times.
SECTION: Store Tissue
43. Transfer sample slide/s to a coplin jar containing 50 mls of CODEX® Storage Buffer and store at 4°C until imaging.