Procedures

1. Immobilize stage 11 embryos or juveniles in ice-cold relaxant.

2. Fix the embryo/juvenile in cold GPA fixative at 4°C for 3 hours.

3. Wash with 70% ethanol for several times.

4. Wash with PBS for 3 times.

5. Treat the sample with 0.5 mg/ml collagenase (type IV) in PBS for 30 minutes at room temperature.

6. Wash with 1% sodium metabisulfite in PBS for 3 times.

7. Treat the sample with reducing buffer.

8. Wash with PBS + 1% Triton X-100 for several times and then proceed to the blocking step of standard immunostaining protocol.

Recipes

• GPA fixative: 6.25% glutaraldehyde, 75% saturated picric acid, 5% glacial acetic acid (25% glutaraldehyde : saturated picric acid : glacial acetic acid = 50 : 150: 1), supplemented with 1% sodium metabisulfite

• Reducing buffer: 1% sodium borohydride (cautious! highly reactive!), 1% sodium metabisulfite in PBS