Double in situ hybridization is performed by hybridizing two different probes (digoxigenin and fluorescein) labeled at the same time. One antibody and chromogenic substrate are used for each. The 1st antibody is inactivated after staining and then second antibody and chromogenic substrate are used. Alternatively, fluorescent substrates can be used. Used on post-implantation embryos.

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1. Dissect embryos out in PBS, then fix in 4% paraformaldehyde, rocking for 1 h. (RT) to overnight (+4° C).
2. Wash in PBT (PBS, 0.1% Tween 20), 2 times for 5 min.
3. Dehydrate through 25%, 50%, 75% and twice 100% methanol in PBT, 5 min. for each step [or directly in 100% methanol]l. Embryos are stored in methanol 100% at -20o C.

4. Rehydrate through 75%, 50%, 25% methanol in PBT and twice in PBT, 5 min. each step.
5. Bleach in freshly-made 6% hydrogen peroxide in PBT, 1h.
6. Wash 2 to 3 times in PBT for 5 min.
7. Treat with 10 ug/ml proteinase K in PBT (room temp). Duration of the treatment depends on the embryonic stage and the batch of proteinase K, and should be optimized (the stock of 10mg/ml should be aliquoted and frozen,as the enzyme self-digests, and loses activity). A typical incubation time is about, 5 min for 8.5 dpc embryos, 8-10 min for 9.5 dpc embryos.
8. Stop the digestion with 2 mg/ml of glycine in PBT. Glycine stocks can be made at 100mg/ml and stored frozen. Do not re-freeze.
For small embryos, proteinase K/glycine step can be replaced by a RIPA detergent treatment (150 mM NaCl, 1% Nonidet P-40, 0.5% Na deoxycolate, 0.1% SDS, 1 mM EDTA, 50 mM Tris pH 8.0), 3 times 30 min. each.

9. Wash twice 5 min with PBT.
10. Fix in freshly prepared 0.2% glutaraldehyde+4% paraformaldehyde in PBS or PBT for 20 min.
11. Wash in PBT, 2 times 5 min.
1. Prehybridize at least 1 h at 65° C with hybridization buffer: 50% deionized or fresh formamide, 5X SSC, 1% SDS, 100 mg/ml tRNA, 50 mg/ml heparin (50 mg/ml stock in 4X SSC). CHAPS can be added at 0.1% final to avoid sticky embryos.
Embryos can be stored after this step at -20° C.
13. Replace with hybridization buffer containing 1 mg/ml digoxygenin- and/or fluorescein-labelled RNA probes. Incubate at 65° C overnight.

14. Wash 2 times for 30 min at 65° C with: 50% formamide, 5X SSC, 1% SDS.
15. Wash 2 times for 5 min with RNase A buffer (0.5 M NaCl, 10 mM Tris pH 7.5, 0.1% Tween 20).
16. Incubate with 100 ug/ml RNase A in its buffer at 37° C for 1 h.
Steps 15 to 17 can be skipped for small embryos (< 9.5 dpc)

17. Wash 5 min with 50% formamide, 2X SSC.
18. Wash 20 min at 65° C with 50% formamide, 2X SSC.
19. Wash 2 times 5 min with TBST (0.14 M NaCl, 2.5mM KCl, 25 mM Tris pH 7.5, 0.1% Tween 20) or PBT .
20. Incubate embryos with 1% blocking reagent (Roche) in TBST (or PBT) for at least 1 h.
21. Replace with 1% blocking reagent in TBST (or PBT) containing anti-digoxigenin or anti-fluorescein antibody conjugated with alkaline phosphatase (1:1000, Roche). To inhibit endogenous alkaline phosphatase, 2 mM levamisole can be added(we don’t on embryos < 9.5 dpc). Incubate overnight at +4° C.

22. Wash several times in TBST (depends on the size of the embryos). Start with brief washes (15 min) then extend the time for the last washes (1 hour to overnight).For smaller embryos(up to 8.5 dpc), 6 times 30 min should be sufficient.
23. Wash 2 times 10 min with NTMT (100 mM NaCl, 100 mM Tris pH 9.5, 50 mM MgCl2, 0.1% Tween 20, freshly prepared).
24. Stain purple or blue :
Purple: 4.5 ml/ml NBT (75 mg/ml in 70% dimethylformamide), BCIP 3.5 ml/ml (50 mg/ml in dimethylformamide) in NTMT.
Blue: 1.5 ml/ml K ferricyanide (0.1M), 3 ml/ml K ferrocyanide (0.1 M), BCIP 3.5 ml/ml (50 mg/ml in dimethylformamide) in NTMT at 37°C.
25. Wash 3 times for 5 min with PBT.
NB: there is another AP substrate staining solution from Boehringer (Roche) called BM Purple. Do not use tween in the buffer if using this stain.
26. Incubate 10 min in 0.1M glycine-HCl pH 2.2, 0.1% Tween 20 to remove the first antibody.
27. Wash 2 times for 5 min with PBT.
28. Wash 2 times 5 min with TBST.
29. Repeat blocking and washing steps, starting from step 20.

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PROBE PREPARATION(for 20 ul reaction)
1 ug of linearized DNA
2 ul Transcription buffer 10X(Roche)
1 mM ATP, GTP and CTP (each)
0.65 mM UTP
0.35 mM Digoxigenin-11-UTP (Roche)
20-50 units of RNase inhibitor
20 units of RNA polymerase

Incubate at 37° C for 2 hours.
Run 2 ml on a gel to quantify RNA (compare to DNA band)
Stop reaction by adding 2 ml of EDTA 0.2 M
Precipitate with 2.5 ml of 4M LiCl and 75 ml of EtOH, 20 min at -70° C.
-Wash with EtOH 70%.
Resuspend in 50 ml of water.
We generally use 5 to 10 ul of probe per ml of hybridization buffer.