Gal Staining

1. Dissect testes in testis buffer or TBI (see Note 2). Transfer to a 24-well tissue culture dish or glass staining block (see Note 17).

2. Fix in 1% glutaraldehyde (wear gloves) in PBS for 15 min (see Note 18).

3. Rinse three times in X-gal buffer. Leave in this buffer for at least 30 min.

4. Prepare 5 mL of staining solution and warm to 37°C.

6. Incubate tissue in staining solution plus X-Gal at 37°C for 1 h to overnight. Monitor the reaction and leave until color develops.

7. Wash in X-gal buffer, counterstain (if desired) by incubating in 1 |g/mL Hoechst 33258 for 15 min, and mount in 85% glycerol.

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