TITLE: Phenocycler-Fusion Staining Protocol For Bone Marrow Tissue
AUTHORS: Kyung J Ahn, Shovik Bandyopadhyay, Anusha Thadi, Kai Tan

[DESCRIPTION]
This protocol describes the method for antibody staining of FFPE bone marrow tissue on slides using CODEX barcoded antibodies.  The Akoya Phenocycler-Fusion user manual was modified to include photobleaching steps.  Included are the stepwise protocols for tissue procurement, tissue decalcification, tissue sectioning, pre-staining, deparaffinization, antigen retrieval, photobleaching, antibody staining, post-fixation, and tissue storage. 

[GUIDELINES]
Use positively charged slides.
Keep tissue sections from drying during transfer steps

[STEPS]
SECTION: Tissue Pre-treatment
14. Bake sample slide/s in an incubator at 65°C for 1-3 hours until paraffin thoroughly melts.
SECTION: Tissue Deparaffinization and Hydration
16. 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
17. 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
18. Immerse sample slide/s in the coplin jar containing the 1x Citrate buffer and cover the coplin jar with aluminum foil. 
SECTION: Antigen Retrieval
19. 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
20. Set the pressure cooker to the high-pressure protocol and let the tissue incubate for 20 minutes.
SECTION: Antigen Retrieval
21. 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
15. Cool sample slide/s at room temperature for 5 minutes to allow the sample slide/s to cool to room temperature.
SECTION: Wash Tissue
22. 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
23. Place sample slide/s in a second coplin jar filled with ddH2O and incubate for 2 minutes.
24. Place sample slide/s in a third coplin jar filled with 1X PBS and incubate for 2 minutes.
SECTION: Photobleach Tissue
25. 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
26. Sandwich the petri dish between two broad-spectrum LED light sources for 45 minutes at 4°C.
SECTION: Photobleach Tissue
27. 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
28. Wash the sample slide/s three times for 2 minutes each in coplin jars containing 1X PBS.
SECTION: Wash Tissue
29. Immerse the sample slide/s in a coplin jar containing 50 mls of CODEX® Hydration Buffer and incubate for 2 minutes.

SECTION: Wash Tissue
30. 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
31. 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
32. 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
33. 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
34. Add the appropriate volume of each CODEX antibody to the Antibody Cocktail Solution.
SECTION: Preparation of the Antibody Cocktail Solution
35. Pipette to mix and briefly spin down the tube.
SECTION: Tissue Staining
36. Remove sample slide/s from the coplin jar containing Staining Buffer and place it on the tray of a humidity chamber.
SECTION: Tissue Staining
37. Add 195 μL of the Antibody Cocktail to each of the sample slide(s) and ensure that the liquid covers the entire tissue.
SECTION: Tissue Staining
38. Incubate overnight in the humidity chamber at 4°C.
SECTION: Wash Tissue
39. 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
40. Place sample slide/s in a second coplin jar containing CODEX® Staining Buffer for 2 minutes.
SECTION: Fix Tissue
41. 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
42. 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
43. Transfer sample slide/s to a second coplin jar of 1X PBS and immerse sample slide/s 2-3 times.
SECTION: Wash Tissue
44. 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
45. Transfer sample slide/s to a coplin jar containing Ice cold methanol and incubate for 5 minutes on ice.
SECTION: Wash Tissue
46. 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
47. Transfer the sample slide/s to a second coplin jar of 1x PBS and immerse sample slide/s 2-3 times.
SECTION: Wash Tissue
48. Transfer the sample coverslip to a third coplin jar of 1x PBS and immerse sample slide/s 2-3 times.
SECTION: Fix Tissue
49. 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
50. Remove sample slide/s from the coplin jar and place it on the tray of a humidity chamber.
SECTION: Fix Tissue
51. 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
52. Incubate for 20 mins.
SECTION: Wash Tissue
53. 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
54. Move the sample slide/s to a second coplin jar containing 1x PBS and immerse sample slide/s 2-3 times.
SECTION: Wash Tissue
55. Move the sample slide/s to a third coplin jar containing 1x PBS and immerse sample slide/s 2-3 times.
SECTION: Store Tissue
56. Transfer sample slide/s to a coplin jar containing 50 mls of CODEX® Storage Buffer and store at 4°C until imaging.
SECTION: Tissue Procurement
1. Femoral head bone marrow tissue obtained from total hip arthroplasty surgery was cut coronally in 4-6 mm thick slices by the Anatomical Pathology Lab at Penn Presbyterian Medical Center.  
SECTION: Tissue Procurement
2. A dental biopsy drill was used to harvest 4 mm diameter cylindrical bone marrow tissue samples.
SECTION: Tissue Fixation
3. Bone marrow tissue was fixed in 4% paraformaldehyde at 4°C for 24 hours.
SECTION: Tissue Decalcification
4. Bone marrow tissue was washed 3 times for 30 minutes in PBS at 4°C.
SECTION: Tissue Decalcification
5. Bone marrow tissue was decalcified in 0.5M EDTA pH8 for 8 days at 4°C. The EDTA was changed every 2 days.
SECTION: Tissue Fixation
6. After decalcification, samples were washed 5 times for 1 hour in 1 X PBS at 4°C. 
SECTION: Tissue Fixation
7. Bone marrow tissue was fixed overnight in 4% paraformaldehyde at 4°C.
SECTION: FFPE Blocks
8. Bone marrow tissue was processed and embedded in paraffin by the Children’s Hospital of Philadelphia pathology core. 
SECTION: FFPE Tissue Sectioning
9. Prepare a water bath at 40°C.
SECTION: FFPE Tissue Sectioning
10. Section the bone marrow tissue at a thickness of 5 μm using a microtome.
SECTION: FFPE Tissue Sectioning
11. Place the sectioned tissue in the water bath for a few seconds until the tissue is completely flat and devoid of folds or wrinkles.
SECTION: FFPE Tissue Sectioning
12. Place a slide in the water bath and gently lay the tissue on the slide as it is removed from the water bath. Make sure the tissue section is placed in the accessible imaging area (gray in image below) of the slide.


 

SECTION: FFPE Tissue Sectioning
13. Place the slide on an angled slide holder and let it dry overnight at room temperature.