6.2.1 Whole-mount in situ hybridization (Inohaya version)

FrontPage

6.2.1 Whole-mount in situ hybridization (Inohaya version)

Keiji Inohaya
Tokyo Institute of Technology

6.2.1.1 Reagents

• 4% paraformaldehyde in phosphate-buffered saline (PBS)
• PBST (0.1% Tween-20 in PBS)
• Methanol
• Hydrogen peroxide (6% hydrogen peroxide in PBST)
• Proteinase K (10 µg/ml in PBST)
• 20x saline sodium citrate (SSC), pH6.0
• Hybridization buffer (50% formamide, 0.1% Tween-20, 5 mg/ml torula RNA, 50 µg/ml heparin, 2x SSC pH 6.0),
• Formamide
• Lamb serum (Heat inactivated at 56 ºC for 30min)
• Anti-digoxigenin Fab fragments, coupled to alkaline phosphatase (option: The antibody is preabsorbed in advance by incubating with homogenized medaka embryos at various stages for 3-5 hours at room temperature.)
• NTMT (100 mM NaCl, 100 mM Tris-HCl, pH9.5, 50 mM MgCl2, 0.1% Tween-20)
• 4-nitro blue terazolium chloride (NBT)
• 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)

6.2.1.2 Method

1. Fix embryos with 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4 ºC.
2. The specimens are dechorionated with forceps.
3. Wash 3 times in PBST (0.1% Tween-20 in PBS).
4. Transfer embryos into 100% methanol, and store at –20 ºC. (This step is necessary for permeabilization of embryos. Embryos can be stored in methanol for months.)
5. Wash 3 times in PBST.
6. Bleach the embryos with 6% hydrogen peroxide in PBST for 1 hour or longer at room temperature.
7. Digest with proteinase K (10 µg/ml in PBST) for 5-10 min at 37 ºC.
8. Fix for 20 min with 4% paraformaldehyde in PBST at room temperature.
9. Wash 3 times with PBST.
10. Transfer embryos into hybridization buffer (50% formamide, 0.1% Tween-20, 50 µg/ml tRNA, 50 µg/ml heparin, 2x SSC pH 6.0), and prehybridize at 55 ºC for 90 min or longer.
11. Hybridize overnight with digoxigenin-labeled RNA probe in the hybridization buffer at 55 ºC.
12. Wash 4 times in 50% formamide/2x SSC at 68 ºC (30 min each).
13. Wash in 25% formamide / 2x SSC for 10min at 68 ºC
14. Wash twice in 2x SSC at 68 ºC (10 min each).
15. Wash twice in 0.2x SSC at 68 ºC (20 min each minimum).
16. Wash 3 times in PBST at room temperature (10 min each).
17. Block for at least 90 min with 5% lamb serum in PBST at room temperature.
18. Incubate with anti- DIG Fab-alkaline phosphatase in PBST overnight at 4 ºC.
19. Wash 6 times in PBST at room temperature (30 min each minimum).
20. Wash 3 times in NTMT (100 mM NaCl, 100 mM Tris-HCl, pH9.5, 50 mM MgCl2, 0.1% Tween-20) at room temperature (10 min each).
21. Incubate in NTMT containing 3.5 µl NBT and 3.5 µl BCIP per ml.
22. Stain for appropriate time (30min to overnight) in the dark.
23. Wash several times in PBST to stop developing of staining.
24. Specimens can store in PBST at 4 ºC for months under the dark.

6.2.1.3 References

Inohaya K, Yasumasu S, Ishimaru M, Ohyama A, Iuchi I, and Yamagami K (1995). Temporal and Spatial Patterns of Gene Expression for the Hatching Enzyme in the teleost Embryo, Oryzias latipes. Dev. Biol. 171, 374-385.

Jowett T (1997) ¡ÈTissue in situ hybridization¡É Wiley and Spektrum.

Yasutake J, Inohaya K, Kudo A (2004). Twist functions in vertebral column formation in medaka, Oryzias latipes. Mech. Dev. 121, 883-894.