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Detection of Apoptosis
InSitu Cell Death Detection Kit, Fluoresein
Roche Cat.# 1 684 795

1. Deparaffinize 20minX2. Rehydrate slides to ddH₂O.
   (100% ETOH 2min. 95% ETOH 2min. ddH₂O 2min X2).
2. Wash in 0.1% PBST (3min X2.
3. Proteinase K:
   20ug/ml in PBS for 15 min at RT.
   10mg/ml PK 100 ul
   PBS 50ml
4. Wash with PBST(PBS+0.1% Tween-20).
5. Positive control: Dnase I grade I 1ug/ml in 50mM Tris 7.5, 1mM MgCl2 , 1mg/ml BSA. Incubate for 15 min at RT.
6. 0.1% PBST 5minX2.
7. Incubate with 0.5% PBST( PBS + 0.5% Tween 20) for 30min at RT.
8. Rinse with PBS.
9. 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)
10. Add TUNEL reaction mixture to the slides. Cover with parafilm
11. Incubate at 37°C for 60min in the dark.
12. Wash with PBS.
13. Cover slides with Vectashild mounting medium (Vector).

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