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11.1.3 TUNEL staining

The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method labels fragmented DNA which is a characteristic of the apoptotic cells.

Yuki Nakatani
Tokyo Institute of Technology

11.1.3.1 Reagents

  • In Situ Cell Detection Kit (AP) by Roche
  • 4% paraformaldehyde
  • methanol
  • PBST: PBS + 0.1% Triton X-100
  • 10µg/ml proteinase K in PBS
  • 0.1% sodium citrate, 0.1% Triton X-100 (freshly prepared)
  • Blocking solution: 3% BSA, 5% lamb serum in 0.1M Tris-HCl (pH7.5)
  • AP buffer: 100mM Tris-HCl (pH9.5), 100mM NaCl, 50mM MgCl2, 0.1% Triton X-100, 1mM Levamisole
  • AP staining solution: 100mM Tris-HCl (pH9.5), 100mM NaCl, 50mM MgCl2, 1mM Levamisole, 337.5µg/ml NBT, 175µg/ml BCIP

11.1.3.2 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
  5. Wash with PBST
  6. 10 µg/ml proteonase K treatment at RT for 5 min
  7. PBST wash
  8. Refix in 4% paraformaldehyde at RT for 20 min
  9. Wash with PBST
  10. 0.1% sodium citrate, 0.1% Triton X-100 on ice for 15 min
  11. Wash with PBST
  12. Replace PBST with reaction mixture (solution 1: solution 2 (kit) = 1:9)
  13. Incubate at 37ºC for 1 hr
  14. Wash with PBST
  15. Incubate in blocking solution at RT for 1 hr
  16. Incubate in 1:2000 AP-conjugated anti-fluorescein antibody at 4ºC, O/N
  17. Wash with PBST
  18. Wash with AP buffer
  19. Incubate in AP staining solution until signals are detected
  20. Stop reaction with PBST when signals are detected

11.1.3.3. Reference

Barrallo-Gimeno A, Holzschuh J, Driever W, Knapik EW. (2004). Neural crest survival and differentiation in zebrafish depends on mont blanc/tfap2a gene function. Development 131 (7), 1463-1477.


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Last-modified: 2019-04-04 (Thu) 14:18:41