[[FrontPage]] *6.2.2 In situ hybridization in fins Yuki Nakatani~ Sae Sakaguchi~ Tokyo Institute of Technology **22.214.171.124 Reagents -4% formaldehyde -PBT: PBS + 0.1% Tween-20 -methanol -10µg/ml proteinase K in PBT -Hybridization buffer: 50% formamide, 5xSSC, 0.1% Tween-20, 50µg/ml heparin, 10mg/ml torula RNA -2xSSC, 50%formamide, 0.1%Tween20 -2xSSC: 2xSSC, 0.1% Tween-20 -0.1xSSC: 0.1xSSC, 0.1% Tween-20 -Blocking solution: 5% lamb serum in PBT 1:2000 AP-conjugated anti-digoxigenin (DIG) Fab fragments in blocking solution -AP buffer: 100mM Tris-HCl (pH9.5), 100mM NaCl, 50mM MgCl2, 0.1% Tween-20, 1mM Levamisole -AP staining solution: 100mM Tris-HCl (pH9.5), 100mM NaCl, 50mM MgCl2, 1mM Levamisole, 337.5µg/ml NBT, 175µg/ml BCIP -RNA probes: Probes are synthesized according to the standard procedure using SP6, T7 or T3 RNA polymerase incorporating a DIG substituted ribonucleotide. **126.96.36.199 Method ***Fixation: +Fix wild-type or regenerating fins in 4% paraformaldehyde, 4ºC, O/N. +Leave fixed fins at RT for 1 hour. +Wash fins with PBST for several times. +Dehydrate gradually through a methanol series (25-50-75% methanol in PBST) and replace fixation with 100% methanol. +Store in 100% methanol at -20ºC. ***Hybridization: +Remove methanol and serially replace with 75%-50%-25% methanol in PBT. +Wash fins with PBST, 3 times at RT. +Remove PBST and replace with 10 µg/ml proK in PBST. +Incubate at 37ºC for 10 min. +Wash with PBST. +Refixed in 4% paraformaldehyde at RT for 20 min. Do not incubate more than 20 min maximum. +Wash with PBST. +Wash with 0.1M triethanolamine-HCl (pH8.0) at RT for 5 min, twice. +Incubate in 0.25% acetic anhydrate in 0.1M triethanolamine-HCl (pH8.0) at RT for 10 min. +Wash with PBST. (option: Rinse with hybridization buffer.) +Pre-hybridization: at least 1 hour at 65ºC in hybridization buffer. +Hybridization: O/N, 65ºC in hybridization buffer containing probe (probe concentration=1 µg/ml). If required, preheat a probe at 80ºC for 10 min, and then put on ice for at least 3 min before adding to the hybridization buffer. ***Washing: +Remove hybridization buffer and replace with 2xSSC, 50% formamide, 0.1% Tween20. +Incubate at 65ºC for 30 min. +Repeat again. +Wash the fins with 2xSSC at 65ºC for 15 min. +Wash the fins with 0.1xSSC at 65ºC for 30 min, three times. +Wash the fins with PBST at RT for 5 min, twice. Blocking and antibody staining: +Incubate in blocking solution at RT for at least 1 hour. +Replace blocking solution with 1:2000 (up to 1:6000) AP-conjugated anti-DIG Fab fragments and incubate at 4ºC for overnight. Clear signals could be detected using recycled antibodies. ***Staining: +Wash the fins with PBST at RT for 15 min, six times. Longer incubation would be suggested to minimize the background staining. +Wash with AP buffer at RT for 5 min, twice. +Incubate the fins in AP staining solution at RT until signals are detected. +The reaction is stopped by dilution in PBST. **188.8.131.52 Reference Katogi R, Nakatani Y, Shin-I T, Kohara Y, Inohaya K, Kudo A. (2004). Large-scale analysis of the genes involved in fin regeneration and blastema formation in the medaka. Olyzias latipes. Mech Dev. 121, 861-872.