TUNEL Reaction

  1. Fix in 1% paraformaldehyde in PBS, pH 7.4 for 10 min. at room temp.
  2. Wash 2x with PBS.
  3. Post-fix in pre-cooled EtOH: Acetic acid 2:1 for 5 min. at -20°C.
  4. Rinse 2x with PBS.
  5. Gently tap off excess liquid and carefully blot or aspirate.
  6. Incubate 1 hour in 50ul equilibration buffer (Apoptag kit) at room temp.
  7. Incubate in 150ul of the TdT mix (100ul reaction buffer and 50ul TdT enzyme) at 37°C for 1 hour.
  8. Stop reaction in 34:1 ration of H2O: stop buffer (apoptag), agitate for 15 sec. and incubate for 10 min. R.T. Wash 3x in PBT for 5 min.
  9. Incubate with anti-DIG antibody (Rhodamine conjugated, provided by vendor) for 2 hrs at room temp.
  10. Wash 3x with PBT.
  11. Wash with PBT and mount.

Materials: Apoptag from Chemicon S7165 (Rhodamine)