Application suite advanced

DNA fragmentation was examined using a TUNEL assay, which preferentially labels DNA strand breaks generated during apoptosis; specifically, the Fluorescein In situ Cell Death Detection kit (cat. no. 11684809910; Roche Applied Science) was used according to the manufacturer's protocol. The 5-µm thick tissue sections of ascending aorta obtained from paraffin blocks, prepared as aforementioned, were dewaxed in xylene and rehydrated in decreasing alcohol steps (ethanol at 100, 96, 80, 50% and distilled water). Then, the sections were incubated with proteinase K working solution at 37°C for 30 min for permeabilization and subsequently rinsed twice with PSB. Thereafter, the sections were treated with the TUNEL reaction mixture for 60 min at 37°C in the dark and rinsed three times with PBS. The nuclei were stained with Hoechst 33342 solution (1:1,000 in PBS; cat. no. 62249; Thermo Fisher Scientific, Inc.) for 15 min at room temperature. Finally, the coverslip was applied using PBS as the mounting medium and the stained sections were examined by a confocal microscope (Leica Confocal Microscope TCS SP8; Leica Microsystems, Inc.) and analyzed by Leica Application Suite advanced fluorescence software version 3.3.0. (Leica Microsystems, Inc.), and images of five random and non-overlapping fields were examined at an objective magnification of ×200 for analysis.