TITLE: Immunofluorescence staining ASE, vibratome sections
AUTHORS: eduard.bentea, María Sanchiz Calvo, Veerle Baekelandt

[DESCRIPTION]
Protocol for performing immunofluorescence staining using antibody signal enhancement (ASE) on vibratome cut brain sections from rats or mice.

[STEPS]
SECTION: Day 1
1. Briefly rinse sections in 1/2 PBS + 1/2 AD and mount on Superfrost Plus Slides.
SECTION: Day 1
2. Air dry slides overnight at room temperature.
SECTION: Day 2
3. 1X PBS rinse.
SECTION: Day 2
4. Note: All washes performed in Tissue-Tek Staining Trays, on the wobbler. 100 mL / tray.
SECTION: Day 2
5. Antigen retrieval step (using Steamer).
SECTION: Day 2
5.1. Fill water to the maximum level in the steamer (use distilled water).
SECTION: Day 2
5.2. Place Tissue-Tek Staining Trays containing Antigen retrieval solution (Tris-HCl-EDTA buffer pH 9.0 + 0.05% SDS ; see recipe in Materials) in the steam bowl. Fill with 100 mL / tray.
SECTION: Day 2
5.3. Wait at least 15 min for the solutions in the steam bowl to reach 95-98oC.

SECTION: Day 2
5.4. Place the glass slides with the tissue in the Antigen retrieval solution.
SECTION: Day 2
5.5. Start timer for antigen retrieval (30 min).
SECTION: Day 2
5.6. Refill with distilled water the tank as needed.
SECTION: Day 2
6. After antigen retrieval : Place the Tissue-Tek Staining Trays from the Steamer on ice for 20 min (in cold room).

SECTION: Day 2
7. Dry slides and add hydrophobic barriers on each side of the tissue section. Do not let the barrier touch the tissue. 
SECTION: Day 2
8. Wash slides with ASE Wash buffer (PBS + 0.5% Tween-20 ; see recipe in Materials) for 3 min at room temperature on wobbler.

SECTION: Day 2
9. Wash slides with ASE Wash buffer for 3 min at room temperature on wobbler.

SECTION: Day 2
10. Prepare box for slide incubation. Place wet tissue paper on the bottom to create a humid chamber. Add the glass slides inside facing up.
SECTION: Day 2
11. Pipette 500 µL ASE Blocking solution (PBS + 2% donkey serum , 50 mM glycine, 0.05% Tween-20, 0.1% Tergitol, 0.1% BSA ; see recipe in Materials) per glass slide.

SECTION: Day 2
12. Block for 30 min at room temperature.

SECTION: Day 2
13. Discard the blocking solution, and pipette instead 500 µL of primary antibody solution per glass slide. Dilute primary antibodies in ASE primary antibody buffer (PBS + 10 mM glycine, 0.05% Tween-20, 0.1% Tergitol, 0.1% H2O2 ; see recipe in Materials).

SECTION: Day 3
15. Wash slides with ASE wash buffer (quick rinse).

SECTION: Day 3
16. Wash slides with ASE wash buffer for 3 min at room temperature on wobbler.

SECTION: Day 3
17. Wash slides with ASE wash buffer for 3 min at room temperature on wobbler.
SECTION: Day 3
18. Place the glass slides in the incubation box. Pipette 500 µL of secondary antibody solution per glass slide. Dilute secondary antibodies in ASE secondary antibody buffer (PBS + 0.1% Tween-20 ; see recipe in Materials).

SECTION: Day 3
19. Incubate with secondary antibodies in the dark for 120 min at room temperature.

SECTION: Day 3
20. Wash slides with PBS (quick rinse).
SECTION: Day 3
21. Wash slides with PBS for 5 min at room temperature on wobbler.

SECTION: Day 3
22. Wash slides with PBS for 5 min at room temperature on wobbler.
SECTION: Day 3
23. Remove hydrophobic barriers. Allow sections to dry and mount with Mowiol.
SECTION: Day 2
14. Incubate with primary antibodies overnight at 4oC.