11.1.3 TUNEL staining

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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.