2.5. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining

Sections of 5–6 μm thickness obtained from paraffin blocks were mounted on polylysine glass slides. Apoptotic cells were detected using the ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon International, Temecula, CA, USA, Cat no: S7101) according to the manufacturer’s instructions. Tissues deparaffinized with xylene were passed through graded alcohol series and washed with PBS. Tissues incubated for 10 min with 0.05% proteinase K were incubated for 5 min with 3% hydrogen peroxide to prevent endogenous peroxidase activity. After washing the tissues with PBS, they were incubated with equilibration buffer for 6 minutes and incubated for 60 min with a working solution (70% μL reaction buffer + 30% TdT enzyme) at 37 ºC. Apoptotic cells were visualized with the diaminobenzidine (DAB) substrate. The cross-staining sections with Harris hematoxylin were sealed with the appropriate capping solution. The prepared preparations were examined, photographed, and examined under computer‐assisted Leica DM500 light microscope (Leica Microsystems, Wetzlar, Germany). In the evaluation of TUNEL staining, blue‐stained nuclei by Harris hematoxylin were evaluated as normal, whereas cells displaying brown-stained nuclei were considered as apoptotic. At least 500 cells were counted in normal and apoptotic areas in randomly selected areas at × 10 magnification in sections. Apoptotic index (AI) was calculated as a percentage (%) by proportioning apoptotic cells to total (normal + apoptotic) cells [20].