TUNEL Protocols
Fixing cells
1. Wash cells on slides.
2. Fix cells with 2-4% paraformaldehyde in PBS for 15 minutes.
3. Wash the cells in PBS three times for 5 minutes.
4. Permeabilize cells with 0.2% Trion X-100 in PBS for 5 minutes at room temperature.
5. Wash the cells in PBS three times for 5 minutes.
Fluorescein labeling
6. Equilibrate in the equilibration buffer (200 mM potassium or sodium cacodylate (pH 6.6), 25 mM Tris-HCl (pH6.6), 0.2 mM DTT, 0.25 mg/ml BSA, 2.5 mM cobalt chloride).
7. Make a labeling mix: 90 ml equilibration buffer, 10 ml nucleotide mix (50 mM fluorescein-12-dUTP, 100 mM dATP, 10 mM Tris-HCl (pH7.6), 1 mM EDTA), and 2 ml TdT enzyme (20 units).
8. Add 50-100 ml labeling mix to each slide, cover with a plastics cover slide. Incubate at 37 °C for 1 hour in a dark humidified chamber.
9. Wash the cells in 2X SSC three times for 10 minutes each in dark.