TUNEL Assay for Apoptosis
(for paraffin embedded sections)
- Xylene: 10 minutes (3 changes)
- Absolute ethanol :5 minutes (2 changes)
- 95% ethanol :5 minutes.
- 70% ethanol :10 minutes.
- Wash in PBS (pH 7.4): 3 minutes (3 changes)
- Apply protein digesting enzyme (20ug/ml)
to tissues for 15 minutes, room temperature.
- Wash in ddH2O for 5 minutes (3 changes)
- 2% H2O2 in PBS for 5 minutes.
- Wash
in PBS for 5 minutes.
- Apply 2 drops (about 60 ul) equilibration buffer and put on plastic coverslip for 1 minute.
- Put 50 ul od TdT
onto section. Apply plastic coverslip and incubate in humidified chamber at 37 centigrade for 1 hour.
- Put section in stop/wash buffer
and incubate at 37 centigrade for 30 minutes.
- Wash with PBS 5 minutes (3 changes)
- Apply 2 drops (about 50 ul) anti-digoxigenin-peroxidase
and put on plastic coverslip at room temperature for 30 minutes.
- Wash in PBS 5 minutes (4 changes)
- H2O2/DAB (0.05% DAB with 0.04%H2O2) for 6 minutes.
- Wash in ddH2O.
- 0.5% methyl green 3-5 minutes.
- Wash in ddH2O 1 minute (3 changes).
- Rinse in butanol 30 seconds
(3 changes).
- Xylene 3 minutes (3changes).
- Mount the sections with coverslip.