Deadend colorimetric tunel system detection kit

Brain sections were also examined using the DeadEnd colorimetric TUNEL system detection kit (Promega, Madison, Wisconsin, USA). First, sections were deparaffinized with xylene, rehydrated in a graded ethanol series, and rinsed for 15 min in 0.1 M PBS. Second, sections were treated with 20 μg/ml of proteinase K for 20 min at room temperature. Then, sections were treated with 3% (v/v) H₂O₂ in methanol for 20 min to inactivate endogenous peroxidase. After washing with PBS for 15 min, sections were incubated in the labeling reaction mixture containing terminal deoxynucleotidyl transferase and the deoxynucleotides at 4°C overnight. After incubation, all the sections were rinsed in PBS for 15 min and incubated with horseradish peroxidase (1 : 500) for 30 min at room temperature. Then, sections were washed extensively with PBS for 15 min and treated with DAB solution (30 mg of DAB and 200 μl of H₂O₂/100 ml of PBS) for 10 min at room temperature in the dark. After washing under running water, all the sections were counterstained with H&E for 30 s. Finally, sections were dehydrated in an increasing graded ethanol series, cleared in xylene, and mounted with a cover slip. By TUNEL staining, the apoptotic nuclei were identified by the presence of dark brown staining.