Detection of Apoptosis
InSitu Cell Death Detection Kit, Fluoresein
Roche Cat.# 1 684 795
- Deparaffinize 20minX2. Rehydrate slides to ddH2O.
(100% ETOH 2min. 95% ETOH 2min. ddH2O 2min X2).
- Wash in 0.1% PBST (3min X2.
- Proteinase K:
20ug/ml in PBS for 15 min at RT.
10mg/ml PK 100 ul
- Wash with PBST(PBS+0.1% Tween-20).
- Positive control: Dnase I grade I 1ug/ml in 50mM Tris 7.5, 1mM MgCl2 , 1mg/ml BSA. Incubate for 15 min at RT.
- 0.1% PBST 5minX2.
- Incubate with 0.5% PBST( PBS + 0.5% Tween 20) for 30min at RT.
- Rinse with PBS.
- Prepare TUNEL reaction mixture:
Take 100ul label solution from bottle 2 as negative control.
Add total volume of bottle 1 (50ul) to bottle 2 (450ul) Mix well.
(TUNEL reaction mixture)
- Add TUNEL reaction mixture to the slides. Cover with parafilm
- Incubate at 37°C for 60min in the dark.
- Wash with PBS.
- Cover slides with Vectashild mounting medium (Vector).