6.4.1 Antibody staining in fins

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6.4.1 Antibody staining in fins

Yuki Nakatani
Sae Sakaguchi
Tokyo Institute of Technology

6.4.1.1 Reagents

• 4% paraformaldehyde
• PBST: PBS + 0.1% Triton X-100
• methanol
• 2xMAB: 0.2M maleic acid, 0.3M NaCl, adjust pH to 7.5
• 1xMAB
• 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

6.4.1.2 Method

1. Fins are fixed with 4% paraformaldehyde at 4ºC, O/N.
2. Wash with PBST.
3. Dehydrate gradually through a methanol series (25-50-75-100% methanol in PBST).
4. Store in 100% methanol at -20ºC (several minutes to hours¡Ä).
5. Rehydrate gradually through PBST.
6. 1xMAB wash at RT for 5 min.
7. 0.1% TritonX-100/1xMAB at 4ºC for 15 min, three times.
8. MABD at 4 oC for 30 min, two times.
9. MABDN at 4 oC for 30 min.
10. primary antibody (ex. alpha-acetylated tubulin: 1/1000) in MABDN.
11. 4ºC, O/N or RT for 2 hours with rotate.
12. MABD at 4ºC for 5 min, three times.
13. MABDN at 4ºC for 30 min, four times.
14. secondary antibody (ex. coupled to FITC or Alexa 488) in MABDN.
15. 4ºC, O/N or RT for 2 hours in the dark with rotate.
16. MABD at 4ºC for 5 min in the dark, three times.
17. MAB at 4ºC for 30 min in the dark, four times.
18. Mount and observation.