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*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

+ Fix embryos with 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4 ºC. 
+ The specimens are dechorionated with forceps.
+ Wash 3 times in PBST (0.1% Tween-20 in PBS).
+ 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.)
+ Wash 3 times in PBST.
+ Bleach the embryos with 6% hydrogen peroxide in PBST for 1 hour or longer at room temperature.
+ Digest with proteinase K (10 µg/ml in PBST) for 5-10 min at 37 ºC.
+ Fix for 20 min with 4% paraformaldehyde in PBST at room temperature. 
+ Wash 3 times with PBST.
+ 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.
+ Hybridize overnight with digoxigenin-labeled RNA probe in the hybridization buffer at 55 ºC. 
+ Wash 4 times in 50% formamide/2x SSC at 68 ºC (30 min each).
+ Wash in 25% formamide / 2x SSC for 10min at 68 ºC
+ Wash twice in 2x SSC at 68 ºC (10 min each).
+ Wash twice in 0.2x SSC at 68 ºC (20 min each minimum).
+ Wash 3 times in PBST at room temperature (10 min each).
+ Block for at least 90 min with 5% lamb serum in PBST at room temperature.
+ Incubate with anti- DIG Fab-alkaline phosphatase in PBST overnight at 4 ºC. 
+ Wash 6 times in PBST at room temperature (30 min each minimum).
+ 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). 
+ Incubate in NTMT containing 3.5 µl NBT and 3.5 µl BCIP per ml.
+ Stain for appropriate time (30min to overnight) in the dark.
+ Wash several times in PBST to stop developing of staining. 
+ 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.
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