Immunohistochemistry Protocol

Make up 1L of fresh Phosphate Buffered Saline (PBS) from 10x stock solution.

Make up PBS-T: 0.2–0.5% Triton X-100 in PBS (stir 100-250mg Triton in 50mL of PBS).

Place each series in a glass vial and rinse mouse brain sections with PBS 3 times before starting primary reactions.

Add 1mL of PBS-T with 2% Normal Donkey Serum (20µL) to each series and swirl briefly.

Optional blocking step : leave slices in PBS-T and 2% Normal Donkey Serum at Room temperature for 45–60 min.

Add primary antibody to each series.

Shake gently for 48h 0m 0s at 4°C (sections should barely revolve around the vial).

Rinse sections with PBS 3 times before starting secondary reactions.

Add 1mL of PBS-T with 2% Normal Donkey Serum (20µL) to each series and swirl briefly.

Add secondary antibody to each series.

Shake gently for 1h 30m 0s at Room temperature, protected from light (sections should barely revolve around the vial).

Rinse sections with PBS 3 times before mounting.

Mount sections serially on slides with Prolong Diamond Anti-fade mounting media; protect slides from light and keep at 4°C after 24h 0m 0s drying at Room temperature.