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Star Republic: Guide for Biologists

TUNEL

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 (Promega)

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.

biotin labeling

6a. Equilibrate with 200 ml of 1X TdT buffer + 1 mM Cobalt Chloride for 5 minutes.

7a. Incubate in 100 ml of the labeling mix (1X TdT buffer, 1 mM Cobalt Chloride, 10 mM BrdUTP, 250 units TdT enzyme/ml) at 37 C for 1 hour in a humidified chamber.

8a. Wash in PBS for 3 times, 5 minutes each.

9a. Add 200 ml FITC-avidin or Texas red-avidin (1:100) in 4X SSC/0.2% BSA.

10a. Incubate at 37 C for 1 hour in a dark humidified chamber.

11a. Wash the cells in 4X SSC for 5 minutes in dark.

BrdU labeling

6b. Equilibrate with 200 ml of 1X TdT buffer (0.2M potassium or sodium cacodylate, 25mM TRIS-HCl, pH 6.6, 0.25mg/ml BSA) + 1 mM cobalt chloride for 5 minutes.

7b. Incubate in 100 ml of the labeling mix (1X TdT buffer, 1 mM cobalt chloride, 2.5 mM biotin-dATP, 250 units terminal transferase TdT enzyme/ml) at 37 C for 1 hour in a humidified chamber.

8b. Rinse 3X in PBS.

9b. Add diluted anti-BrdU-FITC antibody and incubate at room temperature for 1 hour in the dark.

10b. Wash cells 3X in PBS in the dark.

Nuclei staining

12. Stain cells in 1 ml/ml propidium iodide or 10 mg/ml DAPI in PBS. Or mount directly in VECTASHIELD + DAPI. RNase may be included in the nuclei staining to eliminate staining for RNA.