Immunofluorescence staining ASE, vibratome sections

Briefly rinse sections in 1/2 PBS + 1/2 AD and mount on Superfrost Plus Slides.

Air dry slides overnight at room temperature.

1X PBS rinse.

Note: All washes performed in Tissue-Tek Staining Trays, on the wobbler. 100 mL / tray.

Antigen retrieval step (using Steamer).

Fill water to the maximum level in the steamer (use distilled water).

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.

Wait at least 0h 15m 0s for the solutions in the steam bowl to reach 95-98^oC.

Place the glass slides with the tissue in the Antigen retrieval solution.

Start timer for antigen retrieval (0h 30m 0s).

Refill with distilled water the tank as needed.

After antigen retrieval : Place the Tissue-Tek Staining Trays from the Steamer on ice for 0h 20m 0s (in cold room).

Dry slides and add hydrophobic barriers on each side of the tissue section. Do not let the barrier touch the tissue.

Wash slides with ASE Wash buffer (PBS + 0.5% Tween-20 ; see recipe in Materials) for 0h 3m 0s at room temperature on wobbler.

Wash slides with ASE Wash buffer for 0h 3m 0s at room temperature on wobbler.

Prepare box for slide incubation. Place wet tissue paper on the bottom to create a humid chamber. Add the glass slides inside facing up.

Pipette 500µL 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.

Block for 0h 30m 0s at room temperature.

Discard the blocking solution, and pipette instead 500µL 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).

Incubate with primary antibodies overnight at 4^oC.

Wash slides with ASE wash buffer (quick rinse).

Wash slides with ASE wash buffer for 0h 3m 0s at room temperature on wobbler.

Wash slides with ASE wash buffer for 0h 3m 0s at room temperature on wobbler.

Place the glass slides in the incubation box. Pipette 500µL of secondary antibody solution per glass slide. Dilute secondary antibodies in ASE secondary antibody buffer (PBS + 0.1% Tween-20 ; see recipe in Materials).

Incubate with secondary antibodies in the dark for 2h 0m 0s at room temperature.

Wash slides with PBS (quick rinse).

Wash slides with PBS for 0h 5m 0s at room temperature on wobbler.

Wash slides with PBS for 0h 5m 0s at room temperature on wobbler.

Remove hydrophobic barriers. Allow sections to dry and mount with Mowiol.