1. Replace the PBT after the posthybridization washes with blocking solution (5% sheep serum, 2 mg/mL BSA, 1% DMSO in PBT).
2. Incubate for at least 60 min.
3. Incubate in 1:5000 dilution (0.15 U/mL) of sheep antifluorescein-AP Fab fragments (Boehringer Mannheim) in blocking solution overnight at 4°C.
5. Equilibrate with 100 mM Tris-HCl, pH 8.2, 0.01% Tween-20 at room temperature by washing 3 x 5 min.
6. Stain embryos with Fast Red™ (Boehringer Mannheim).
8. Inactivate the alkaline phosphatase activity by incubating in 100 mM glycine, pH 2.2, 0.1% Tween-20 for 2 x 15 min at room temperature with gentle shaking. Do not incubate in acidic glycine for longer than is necessary, since prolonged incubation reduces the second signal presumably by masking the second antisense probe.
9. Wash four times for 5 min with PBT.
10. Fix in 4% paraformaldehyde in PBS for 20 min.
11. Wash five times for 5 min with PBT.
12. Incubate embryos in blocking solution for 60 min.
13. Incubate in a 1:5000 dilution (0.15 U/mL) of sheep antidigoxigenin-AP Fab fragments (Boehringer Mannheim) in blocking solution overnight at 4°C.
14. Wash in 0.5% Triton X-100 in PBS for 2 h. Note that either DMSO or Tween-20 in the wash solutions causes the final ELF crystals to be large, so they are substituted with Triton X-100.
15. Wash 3 x 5 min at room temperature with the ELF™ prereaction buffer (150 mM NaCl, 30 mM Tris-HCl, pH 7.5).
16. For staining, incubate in 1:100 dilution of the ELF™ substrate in ELF substrate buffer (provided with the ELF™ kit) at room temperature for 5 h to overnight (see Note 18).
17. Monitor the staining reaction using a UV fluorescent microscope with a DAPI filter set at intervals after starting the reaction.
18. Stop the reaction by washing with 25 mM EDTA, 0.05% Triton X-100 in PBS, pH 7.2.
19. Fix the embryos in 4% paraformaldehyde in PBS for 20 min.
20. Mount the tissue with the special aqueous mounting medium supplied with the kit from Molecular Probes (see Note 19).
21. Specimens should be flat-mounted underneath a coverslip for best resolution. View the Fast Red precipitate with a rhodamine filter set and the ELF precipitate with a DAPI filter set.
22. Caution should be taken in interpreting colocalized fluorescent signals with sequential AP antibodies. If the first enzyme is not completely inactivated by the acid-glycine treatment, then any residual activity may give rise to false-positive signals with the ELF substrate. Therefore, controls should be performed where some embryos are not incubated with the second antibody, but otherwise treated identically with the ELF substrate.
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