Immunostaining of Frozen Sections:

1. Dissect out testes, cut into four pieces and place each into OCT and freeze in de-gassed liquid nitrogen (use a vacuum to pump the gas out of the liquid nitrogen).
2. Cut the sections at –17°C to –19°C in a cryostat and quickly fix in ice cold acetone (for about 15 seconds).
3. Store slides with sections, wrapped individually in foil, at –80°C until use.
4. Bring slides to room in the foil so they do not collect condensation. Fix in 2% paraformaldehyde + 0.03% SDS for 3 min. Move to a second coplin jar with 2% paraformaldehyde (no SDS) for 3 minutes. Wash 3x1min in .4% Photoflo in water. Air dry approx. 15 min.
5. Go through PBS washes as follows:
• 10 min in PBS + 0.4% photoflo
• 10 min in PBS + 0.1% Triton X-100
• 10 min in PBS + 10% ADB for blocking

ADB:

• For 50 ml
• 5 ml of Normal Goat Serum
• 1.5 g BSA
• 125 ml of 20% Triton X-100
• 5 ml of 10X PBS
• make up to 50ml with nano-pure water.