Protocols of 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
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.

TUNEL Protocol

TUNEL Staining Protocol for Apoptosis Detection - Enzyme Method

TUNEL assay

TUNEL procedure for bovine embryos

TUNEL Protocol (Hyde Lab.)

Springer Protocols: Full Text: TUNEL Assay: An Overview of Techniques

Cell Surface Labeling and ‘TUNEL’ Protocol

Springer Protocols: TUNEL and Immunofluorescence double staining.

TUNEL STAINING PROTOCOL

TUNEL Staining Protocol - Enzyme

TUNEL Staining Protocol - Fluorescence

TUNEL Labeling on paraffin section

Detection of Cell Death in Spider Embryos Using TUNEL.

TUNEL Protocol

Detection of TUNEL and GFP.

Analysis of TUNEL Staining by Flow Cytometry to Detect Apoptosis .

References:
TUNEL Apoptotic Cell Detection in Tissue Sections: Critical Evaluation and Improvement.
TUNEL Protocols. The three modalities of TUNEL we tested (laboratory protocol, ApopTag kit, and Boehringer Mannheim kit) yielded no significantly different ...www.jhc.org/cgi/content/full/46/3/327