Double in situ Hybridization

Materials and Reagents

1. Methanol
2. 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP)
3. Nitrotetrazolium blue chloride (NBT)
4. EDTA
5. INT
6. Tris
7. Tween 20
8. Glycine
9. Na₂HPO₄
10. HCl
11. NaCl
12. Sodium Citrate
13. MgCl₂
14. KCl
15. PFA
16. Sheep serum
17. BSA
18. Citric acid
19. Formamide
20. Dechorionate
21. Hybe⁺ buffer (5ml/tube)
22. Torula Yeast RNA (Sigma-Aldrich, catalog number: R6225)
23. Heparin (Sigma-Aldrich, catalog number: H0777 )
24. Lamb Serum (GibcoBRL, catalog number: 16070096 )
25. Fast Red staining buffer (FRSB) [1 M Tris (pH 8.2), 0.1% Tween]
26. Proteinase K (Roche Diagnostics, catalog number: 10165921001 )
27. Fast Red talets (Roche Diagnostics, catalog number: 11496549001 ) or INT/BCIP (Roche Diagnostics, catalog number: 11681460001 )
28. NBT/BCIP (Promega Corporation, catalog number: S3771 )
29. Anti-DIG -AP (Roche Diagnostics, catalog number: 11093274910 )
30. Anti-Fluorescine AP (Roche Diagnostics, catalog number: 11426338910 )
31. NBT/BCIP (see Recipes)
32. 1x PBT (see Recipes)
33. 20x SSC (see Recipes)
34. 10x PBS (see Recipes)
35. 4% Paraformaldehyde (see Recipes)
36. Hybe⁺ buffer (5ml/tube) (see Recipes)
37. Heat Inactivated Lamb Serum (see Recipes)
38. Blocking solution (see Recipes)
39. Staining buffer (see Recipes)
40. Stop solution (see Recipes)
    

Equipment

1. Rotator
2. Nalgene filter
3. Hybridization Incubator
   

Procedure