In situ Hybridization in Zebrafish Embryos

Materials and Reagents

1. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
2. PFA (powder) (Thermo Fisher Scientific)
3. DEPC (Sigma-Aldrich, catalog number: D5758 )
4. Glutaradehyde (Sigma-Aldrich, catalog number: G5882 )
5. Roche anti-DIG AP (Roche Diagnostics, catalog number: 11093274910 )
6. Torula yeast RNA (Sigma-Aldrich, catalog number: R6225 )
7. Heparin (Sigma-Aldrich, catalog number: H0777 )
8. Lamb/Sheep Serum (Thermo Fisher Scientific, catalog number: 16070-096 )
9. Formamide (High purity grade)
10. Methanol
11. Sodium Citrate
12. EDTA
13. NaCl
14. KCl
15. MgCl₂
16. Na₂HPO₄
17. KH₂PO₄ 
18. HCl
19. Tween 20
20. Citric acid
    
21. 1x PBT(made from DEPC H2O) (see Recipes)
22. Pronase (Roche Diagnostics, catalog number: 10165921001 ) (see Recipes)
23. NBT/BCIP color substrate (Promega Corporation, catalog number: S3771 ) (see Recipes)
24. Hybe⁺ buffer (see Recipes)
25. Hybe^- buffer (see Recipes)
26. 20x SSC (see Recipes)
27. 10x PBS (see Recipes)
28. 4% paraformaldehyde/PBS (see Recipes)
29. Blocking solution (see Recipes)
30. Stop solution (see Recipes)
31. Heat inactivated lamb serum (see Recipes)
32. Pre-staining buffer (see Recipes)
    

Equipment

1. Hybridization Incubator
2. 1.5 ml tube
3. 24-well plate
4. Aluminum foil
5. Nalgene filter
   

Procedure