2.11. TUNEL Assay

Apoptotic cell death was evaluated with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Dewaxed and rehydrated sections were digested with 10 μg/mL proteinase-K (Roche) for 30 min at 37 °C and labelled with the in situ Cell Death Detection Kit, TMR red (Sigma, Milan, Italy), according to the manufacturer’s instructions. Prior to the labelling procedure, positive controls were treated with DNase I recombinant (50 U/mL in 50 mM Tris–HCl, pH 7.5, and 1 mg/mL BSA) for 10 min at 25 °C, to induce DNA strand breaks. For negative controls, TDT was omitted from the reaction mixture. All samples were counterstained with DAPI and mounted with ProLong™ Gold Antifade Mountant. Slides were observed under a Nikon Eclipse 600 microscope. Native tissue was used as the control. TUNEL-positive cells were counted, and their numbers are expressed as a percentage of the total cell counted.