[[FrontPage]] *6.2.1 Whole-mount in situ hybridization (Inohaya version) Keiji Inohaya~ Tokyo Institute of Technology **220.127.116.11 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) **18.104.22.168 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. **22.214.171.124 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) °»Tissue in situ hybridization°… 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.