TITLE: Multiplex Labeling with Tyramide Fluorophores (Free-Floating Tissues)-Killinger Lab 2024
AUTHORS: Bryan Killinger, Solji_G_Choi, Tyler_Tittle

[DESCRIPTION]
This protocol details the multiplex labeling of free-floating tissues using tyramide fluorophores in the killinger lab (2024).

[STEPS]
SECTION: Day 1:
1. Wash free-floating tissue (3 x 10 minutes) in dilution media (DM).

SECTION: Day 1:
2. Heat water bath 90 min before the antigen retrieval step.

Human samples: 90 °C -95 °C
Mouse samples: 80 °C -85 °C

SECTION: Day 1:
3. Place the dish containing sodium citrate buffer in the water bath and heat it for 10 min.

a.	Sodium Citrate Buffer,pH 6 (1L):
2.94 g Sodium citrate-Trisodium salt (Dihydrate) in 1000 mL DI water. 
Adjust pH to 6.0.
0.5 mL Tween-20. Mix well.
SECTION: Day 1:
4. Wash the tissues in sodium citrate buffer for 5 min .
SECTION: Day 1:
5. Incubate the tissues in the heated sodium citrate buffer for 30 min.
SECTION: Day 1:
6. Cool down the tissues by placing container in an ice bucket for 15 min. 
SECTION: Day 1:
7. Wash in DM for 10 minutes x 2 times. 

SECTION: Day 1:
8. Endogenous peroxidase inhibition and serum blocking step (60 min incubation): 0.3% H2O2+0.1% Sodium Azide in blocking buffer. 

a. Blocking buffer: 
 
 


b. In 50 mL blocking buffer, add 0.5 mL 30% H2O2 + 0.5 mL10% Sodium Azide.

SECTION: Day 1:
9. Dilute primary antibody in blocking buffer. Incubate 10 min at 4 °C.

SECTION: Day 2:
10. Wash (3 x 10 minutes) in dilution media.
SECTION: Day 2:
11. HRP-Secondary antibody incubation 1:1000 dilution (60 min). 

Solvent is 100 mL DM/1 mL normal serum/1 g BSA.

SECTION: Day 2:
12. Wash (2 x 10 minutes) in dilution media.

SECTION: Day 2:
13. Wash in borate buffer for 10 min.

a. 0.05 Molarity (M) Borate buffer pH 8.5

 
 
Mix well to dissolve completely. 
Adjust to pH 8.5.
SECTION: Day 2:
14. Incubate with tyramide fluorophore (TF) for 30 min while blocking light. 

a.	10 mL Borate buffer + 1 µL H2O2 + 5 µL TF.
SECTION: Day 2:
15. View under the microscope to confirm successful staining. 
SECTION: Day 2:
16. Store in PBS and leave at 4 °C. It can be stored for up to 2 weeks. Otherwise, proceed with the antigen retrieval step.
SECTION: Day 3:
17. Heat water bath 90 min before the antigen retrieval step.

a.	Human samples: 90 °C-95 °C
b.	Mouse samples: 80 °C-85 °C

SECTION: Day 3:
18. Place the dish containing sodium citrate buffer in the water bath and heat it for 10 min.

a.	Sodium Citrate Buffer,pH 6 (1L):
2.94 g Sodium citrate-Trisodium salt (Dihydrate) in 1000 mL DI water. 
Adjust pH to 6.0
0.5 mLTween-20. Mix well.



SECTION: Day 3:
19. Wash the tissues in sodium citrate buffer for 5 min.
SECTION: Day 3:
20. Incubate the tissues in the heated sodium citrate buffer for 30 min.
SECTION: Day 3:
21. Cool down the tissues by placing container in an ice bucket for 15 min. 
SECTION: Day 3:
22. Wash in DM for 10 minutes x 2 times.
SECTION: Day 3:
23. Endogenous peroxidase inhibition and serum blocking step (10 min incubation): 0.3% H2O2+0.1% Sodium Azide in blocking buffer.

a. Blocking buffer: 
 
 
b. In 50 mL blocking buffer, add 0.5 mL 30% H2O2 + 0.5 mL 10% Sodium Azide.

SECTION: Day 3:
24. Dilute primary antibody in blocking buffer. Incubate 10 min at 4 °C.
SECTION: Day 4:
25. Wash (3 x 10 minutes) in dilution media.
SECTION: Day 4:
26. HRP-Secondary antibody incubation 1:1000 dilution (60 min).

a.	Solvent is 100 mL DM/1 mL normal serum/1g BSA.

SECTION: Day 4:
27. Wash (2 x 10 minutes) in dilution media.

SECTION: Day 4:
28. Wash in borate buffer for 10 min.

a. 0.05 Molarity (M) Borate buffer pH 8.5
  
 
  
It takes a while to dissolve completely. 
Adjust to pH 8.5.
SECTION: Day 4:
29. Incubate with tyramide fluorophore (TF) on a shaker for 30 min while blocking light. 

a.	10 mL Borate buffer + 1 µL H2O2 + 5 µL TF.
SECTION: Day 4:
30. View under the microscope to confirm successful staining. 
SECTION: Day 4:
31. DAPI staining (20 min)

a.	1:2000 dilution PBS. Block the light.
SECTION: Day 4:
32. Mount the tissues on a slide, cover the slide with Fluoroshield, and coverslip. Seal with nail polish on all sides of the coverslip.
SECTION: Day 4:
33. When the nail polish is completely dried, view under the microscope. Always protect the slides from light. Slides can be stored at 4 °C.
SECTION: Day 1:
1.1. Wash free-floating tissue for 10 min in dilution media (DM) (1/3).

SECTION: Day 1:
1.2. Wash free-floating tissue for 10 min in dilution media (DM) (2/3).
SECTION: Day 1:
1.3. Wash free-floating tissue for 10 min in dilution media (DM) (3/3).
SECTION: Day 1:
7.1. Wash in DM for 10 min (1/2). 
SECTION: Day 1:
7.2. Wash in DM for 10 min (2/2). 
SECTION: Day 2:
10.1. Wash for 10 min in dilution media (1/3).
SECTION: Day 2:
10.2. Wash for 10 min in dilution media (2/3).
SECTION: Day 2:
10.3. Wash for 10 min in dilution media (3/3).
SECTION: Day 2:
12.1. Wash for 10 min in dilution media (1/2).
SECTION: Day 2:
12.2. Wash for 10 min in dilution media (2/2).
SECTION: Day 3:
22.1. Wash in DM for 10 min (1/2).
SECTION: Day 3:
22.2. Wash in DM for 10 min (2/2).
SECTION: Day 4:
25.1. Wash for 10 min in dilution media (1/3).
SECTION: Day 4:
25.2. Wash for 10 min in dilution media (2/3).
SECTION: Day 4:
25.3. Wash for 10 min in dilution media (3/3).
SECTION: Day 4:
27.1. Wash for 10 min in dilution media (1/2).
SECTION: Day 4:
27.2. Wash for 10 min in dilution media (2/2).