1. Wash three times each for 15 min with TBST.
2. Change TBST hourly, continue to wash with TBST throughout the day, and then leave in a final wash overnight.
3. Wash three times for 15 min with freshly prepared NTMT (pH 9.5).
4. Incubate with NTMT containing 0.25 |g/mL NBT and 0.175 |g/mL BCIP. Wrap in foil and leave at room temperature until desired reaction product intensity is achieved (see Note 14). At this point, for in situ hybridizations using two probes, proceed directly to step 6. For single-color reactions, proceed to step 5.
5. For in situ hybridizations using a single probe, stop the reaction by washing thoroughly in PBT containing 20 mM EDTA or in 1X TBST. Refix in 4% paraformaldehyde prior to storing at 4°C or sectioning. Embryos can also be stored at 4°C in autoclaved 90% v/v glycerol, 1X PBS.
6. For in situ hybridizations using two probes, now rinse several times with TBST.
7. Inactivate alkaline phosphatase by incubating embryos in TBST at 70°C for 30-40 min.
8. Wash three times with TBST.
9. Preblock embryos again with 10% goat serum in TBST for 60-90 min.
10. Meanwhile preabsorb the anti-FITC-AP conjugate as in Subheading 3.5., step 9.
11. Remove the 10% serum from embryos, replace with preabsorbed anti-FITC-AP antibody, and rock overnight (or longer) at 4°C.
12. Repeat steps 1 and 2 above.
13. Wash three times for 15 min with freshly prepared NTMT (pH 8.0).
14. Incubate with NTMT containing fast red TR/naphthol AS-MX. Wrap in foil and leave at room temperature until desired reaction product intensity is achieved (usually two to three times the amount of time required for DIG probes).
15. Stop the reaction as in step 5. Refix in 4% (w/v) paraformaldehyde prior to storing or sectioning.
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