6.4.1 Antibody staining in fins
Tokyo Institute of Technology
- 4% paraformaldehyde
- PBST: PBS + 0.1% Triton X-100
- 2xMAB: 0.2M maleic acid, 0.3M NaCl, adjust pH to 7.5
- MABD: 1xMAB, 10mg/ml BSA, 1% DMSO, 0.1% Triton X-100. Make fresh before use.
- MABDN: 2% FCS (or goat/sheep serum) in MABD
- Fins are fixed with 4% paraformaldehyde at 4ºC, O/N.
- Wash with PBST.
- Dehydrate gradually through a methanol series (25-50-75-100% methanol in PBST).
- Store in 100% methanol at -20ºC (several minutes to hoursí─).
- Rehydrate gradually through PBST.
- 1xMAB wash at RT for 5 min.
- 0.1% TritonX-100/1xMAB at 4ºC for 15 min, three times.
- MABD at 4 oC for 30 min, two times.
- MABDN at 4 oC for 30 min.
- primary antibody (ex. alpha-acetylated tubulin: 1/1000) in MABDN.
- 4ºC, O/N or RT for 2 hours with rotate.
- MABD at 4ºC for 5 min, three times.
- MABDN at 4ºC for 30 min, four times.
- secondary antibody (ex. coupled to FITC or Alexa 488) in MABDN.
- 4ºC, O/N or RT for 2 hours in the dark with rotate.
- MABD at 4ºC for 5 min in the dark, three times.
- MAB at 4ºC for 30 min in the dark, four times.
- Mount and observation.