1. Remove the hybridization solution, and wash embryos twice with prewarmed solution 1 each for 30-60 min at 70°C. For all washes, use an excess of wash solution (e.g., a 20-fold excess) over the approximate volume of the embryonic tissue.
2. Wash with solution 1:solution 2 (1:1) for 10 min at 70°C (see Note 11).
3. Wash three times with solution 2.
4. Wash twice with 100 |g/mL RNase A in solution 2 for 30 min at 37°C.
5. Wash with solution 2 for 10 min and then solution 3 for 5 min.
6. Wash twice with prewarmed solution 3 each for 30 min at 70°C.
7. Wash three times with 1X TBST.
8. Preblock embryos with 10% (v/v) goat serum in TBST for 60-90 min.
9. During preblocking, weigh out 3 mg embryo powder into a microfuge tube, add 0.5 mL TBST, and heat at 70°C for 30 min. Cool on ice, and add 5 ||L goat serum and 1 |L anti-DIG-AP antibody (Boehringer-Mannheim). Shake gently at 4oC for at least 1 h to preabsorb the antibody. Spin in microfuge for 10 min. Dilute the supernatant to 2 mL with 1% goat serum in TBST (see Notes 12 and 13).
10. Remove the goat serum from the embryos, replace with preabsorbed anti-DIG-AP antibody (step 9), and rock overnight at 4°C.
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