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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:

  1. Fix wild-type or regenerating fins in 4% paraformaldehyde, 4ºC, O/N.
  2. Leave fixed fins at RT for 1 hour.
  3. Wash fins with PBST for several times.
  4. Dehydrate gradually through a methanol series (25-50-75% methanol in PBST) and replace fixation with 100% methanol.
  5. Store in 100% methanol at -20ºC.

Hybridization:

  1. Remove methanol and serially replace with 75%-50%-25% methanol in PBT.
  2. Wash fins with PBST, 3 times at RT.
  3. Remove PBST and replace with 10 µg/ml proK in PBST.
  4. Incubate at 37ºC for 10 min.
  5. Wash with PBST.
  6. Refixed in 4% paraformaldehyde at RT for 20 min. Do not incubate more than 20 min maximum.
  7. Wash with PBST.
  8. Wash with 0.1M triethanolamine-HCl (pH8.0) at RT for 5 min, twice.
  9. Incubate in 0.25% acetic anhydrate in 0.1M triethanolamine-HCl (pH8.0) at RT for 10 min.
  10. Wash with PBST. (option: Rinse with hybridization buffer.)
  11. Pre-hybridization: at least 1 hour at 65ºC in hybridization buffer.
  12. 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:

  1. Remove hybridization buffer and replace with 2xSSC, 50% formamide, 0.1% Tween20.
  2. Incubate at 65ºC for 30 min.
  3. Repeat again.
  4. Wash the fins with 2xSSC at 65ºC for 15 min.
  5. Wash the fins with 0.1xSSC at 65ºC for 30 min, three times.
  6. Wash the fins with PBST at RT for 5 min, twice. Blocking and antibody staining:
  7. Incubate in blocking solution at RT for at least 1 hour.
  8. 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:

  1. Wash the fins with PBST at RT for 15 min, six times. Longer incubation would be suggested to minimize the background staining.
  2. Wash with AP buffer at RT for 5 min, twice.
  3. Incubate the fins in AP staining solution at RT until signals are detected.
  4. 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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Last-modified: 2019-04-04 (Thu) 14:18:41