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*6.2.2 In situ hybridization in fins

Yuki Nakatani~
Sae Sakaguchi~
Tokyo Institute of Technology

**6.2.2.1 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.

**6.2.2.2 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.

**6.2.2.3 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.

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