TITLE: Multiplex Labeling with Tyramide Fluorophores for Detecting CIAP-Resistant PSER129 and Proteinase K-Resistant aSyn in situ (Killinger 2024)
AUTHORS: Solji Choi, Bryan Killinger

[DESCRIPTION]
This protocol aims to examine the association of calf-intestinal alkaline phosphatase (CIAP)-resistant alpha-synuclein phosphorylated at serine 129 (PSER129) and proteinase K (PK)-resistant alpha-synuclein (aSyn) in the mouse brain, particularly in M83 transgenic mice treated with preformed fibrils. M83 lines exhibit a notably higher abundance of endogenous PSER129 compared to wild-type mice.

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

Dilution media:
 
 
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
2. Incubate the samples with 1% Triton X-100 in DM for 10 min.

SECTION: Day 1
3. Wash in DM for  10 min.

SECTION: Day 1
4. Wash the tissues in CIAP buffer (2x10 minutes).

CIAP buffer:
 
 
SECTION: Day 1
4.1. Wash the tissues in CIAP buffer for 10 min. (1/2)

SECTION: Day 1
4.2. Wash the tissues in CIAP buffer for 10 min. (2/2)
SECTION: Day 1
5. Incubate the tissues with CIAP at a dilution of 1:333 for 1440 minat 37 °C on a shaker. 

 .
In 500uL CIAP buffer, add 1.5 μl CIAP (30 units). 

SECTION: Day 2
6. Wash in DM (3 x 10 minutes).
SECTION: Day 2
6.1. Wash in DM for 10 min. (1/3)

SECTION: Day 2
6.2. Wash in DM for 10 min. (2/3)
SECTION: Day 2
6.3. Wash in DM for 10 min. (3/3)
SECTION: Day 2
7. Endogenous peroxidase inhibition and serum blocking step (1-hour incubation): 

0.3% H2O2+0.1% Sodium Azide in 50 mL blocking buffer.
Blocking buffer:
 
 
SECTION: Day 2
8. Dilute primary antibody in blocking buffer. Incubate 10 min at 4 °C. 

Recombinant Anti-Alpha-synuclein (phospho S129) antibody (EP1536Y, ab51253), dilution factor: 1:50K
In 30 mLblocking buffer, add 0.6 µL PSER129 antibody.
SECTION: Day 3
9. Wash in DM (3 x 10 minutes).
SECTION: Day 3
9.1. Wash in DM for 10 min. (1/3)

SECTION: Day 3
9.2. Wash in DM for 10 min. (2/3)

SECTION: Day 3
9.3. Wash in DM for 10 min. (3/3)

SECTION: Day 3
10. HRP-Secondary antibody incubation 1:1000 dilution (60 min).  

Solvent is 100 mL DM + 1 mL normal serum + 1 g BSA 

SECTION: Day 3
11. Wash in DM (2 x 10 minutes).
SECTION: Day 3
11.1. Wash in DM for10 min. (1/2)

SECTION: Day 3
11.2. Wash in DM for10 min. (2/2)

SECTION: Day 3
12. Wash in borate buffer for 10 min. 

Borate buffer:
 
 
SECTION: Day 3
13. Incubate with tyramide fluorophore (TF) for 30 min while blocking light.  After this step, always protect the tissues from light. 

Components:
 
 
SECTION: Day 3
15. View under the microscope to confirm successful staining.  
SECTION: Day 3
16. Heat water bath to80 °C-85 °C for  90 min before the primary antibody elution step. 

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

Sodium Citrate Buffer, pH 6.0 (1L):   
 
 
SECTION: Day 3
18. Wash the tissues in sodium citrate buffer for 10 min 

SECTION: Day 3
19. Incubate the tissues in the heated sodium citrate buffer for30 min. 

SECTION: Day 3
20. Cool the dish containing tissues to Room temperature (at least 20 min). 

SECTION: Day 3
21. Wash in DM for 10 min x 2 times.
SECTION: Day 3
22. Mount the tissues on Superfrost Plus Microscope Slides (Fisherbrand), and completely dry it for at least 120 min. 

SECTION: Day 3
23. Heat water bath to 37 °C  for 30 min before the PK digestion step.

SECTION: Day 3
21.1. Wash in DM for 10 min . (1/2)

SECTION: Day 3
21.2. Wash in DM for 10 min . (2/2)
SECTION: Day 3
24. Place the dish containing PBS in the water bath and heat it for 10 min. 

PBS: 
 
 
SECTION: Day 3
25. Add the PK to the PBS at a dilution of 1:666 and mix well. 

 
30 mL PBS, add 45 µLPK.

SECTION: Day 3
26. Incubate the mounted tissues in the PK containing PBS for 30 min. 

SECTION: Day 3
27. Wash the slide in PBS (2 x 5 minutes).
SECTION: Day 3
28. Incubate the slide in 4% PFA for 30 min  at  Room temperature on a shaker.

SECTION: Day 3
29. Wash in DM (2 x 5 minutes).
SECTION: Day 3
30. Block the tissues on slide using Bloxall endogenous blocking solution (Vector Laboratories) for 10 min at  Room temperature on a shaker.

SECTION: Day 3
27.1. Wash the slide in PBS for 5 min. (1/2)

SECTION: Day 3
27.2. Wash the slide in PBS for 5 min. (2/2)

SECTION: Day 3
29.1. Wash in DM for 5 min. (1/2)

SECTION: Day 3
29.2. Wash in DM for 5 min. (2/2)
SECTION: Day 3
31. Dilute primary antibody in blocking buffer. Incubate 10 min at 4 °C. 

Recombinant Anti-Alpha-synuclein antibody (EPR20535, ab212184), dilution factor: 1:20K
In 30 mL blocking buffer, add 1.5 µL aSyn antibody.

SECTION: Day 4
32. Wash in DM (3 x 10 minutes).
SECTION: Day 4
32.1. Wash in DM for 10 min. (1/3)

SECTION: Day 4
32.2. Wash in DM for 10 min. (2/3)
SECTION: Day 4
32.3. Wash in DM for 10 min. (3/3)
SECTION: Day 4
33. HRP-Secondary antibody incubation 1:1000 dilution (60 min).  

Solvent is 100 mL DM + 1 mL normal serum + 1 g BSA 

SECTION: Day 4
34. Wash in DM (2 x 10 minutes).
SECTION: Day 4
34.1. Wash in DM for  10 min. (1/2)

SECTION: Day 4
34.2. Wash in DM for  10 min. (2/2)
SECTION: Day 4
35. Wash in borate buffer for 10 min.

 Borate Buffer:
 
 
SECTION: Day 4
35.1. Incubate with tyramide fluorophore (TF) for 30 min while blocking light.  

SECTION: Day 4
35.2. Wash in DM (2 x 10 minutes).

Wash in DM 10 min. (1/2)

Wash in DM 10 min. (2/2)

SECTION: Day 4
36. View under the microscope to confirm successful staining.  
SECTION: Day 4
37. Wash in PBS (2 x 10 minutes).
SECTION: Day 4
37.1. Wash in PBS for 10 min. (1/2)

SECTION: Day 4
37.2. Wash in PBS for 10 min. (2/2)
SECTION: Day 4
38. Counterstain with DAPI for 20 min at  Room temperature. 

1:2000 dilution in ddH2O or PBS. 

SECTION: Day 4
39. Wash the tissues in PBS (2 x 10 minutes).
SECTION: Day 4
40. Cover the slide with Fluoroshield, and coverslip. Seal with nail polish on all sides of the coverslip. Always protect the slides from light. Slides can be stored at 4 °C.

SECTION: Day 4
39.1. Wash the tissues in PBS for 10 min. (1/2)

SECTION: Day 4
39.2. Wash the tissues in PBS for 10 min. (2/2)
SECTION: Day 3
14. Wash in DM (2 x 10 minutes).
SECTION: Day 3
14.1. Wash in DM 10 min . (1/2)

SECTION: Day 3
14.2. Wash in DM 10 min . (2/2)
SECTION: Day 4
35.3.  Components: