1. Remove slides from box and submerge in 50% formamide, 5X SSC pH 4.5, 1% SDS at 65°C until the coverslips fall off (about 15 min).
2. Wash in 50% formamide 5X SSC pH 4.5, 1% SDS at 65°C, once for 15 min and once for 30 min.
3. Wash three times in 50% formamide, 2X SSC at 65°C; 30 min each.
4. Wash 3 times for 5 min in TBST at room temperature.
5. Block for 30 min at room temperature in blocking buffer.
6. Immerse slides in preabsorbed (see Note 6) alkaline-phosphatase anti-DIG Fab fragments (1/5000 dilution) and incubate at 4°C overnight or for 4 h at room temperature.
7. Wash three times for 20 min in PBST with a rotating stir bar.
8. Wash two times for 5 min in alkaline phosphatase buffer.
9. Incubate in color solution in the dark until desired reaction product intensity is achieved (between 4 h and 10 d).
10. Stop color reaction by incubating for 5 min in 10 mM Tris-HCl pH 7.8, 10 mM EDTA.
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