TUNEL assay

Cell apoptosis in renal tissue was detected using a TUNEL staining kit (Roche Diagnostics). Whole renal tissue was immersed in 30% sucrose solution for 12 h and embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Sakura Finetek USA, Inc.). Subsequently, the frozen renal tissue sections (7-µm-thick) were thawed, dehydrated with a gradient concentration of ethanol, washed with phosphate-buffered saline (PBS), and treated with 15 µg/ml proteinase K (Beyotime Institute of Biotechnology) without DNase. The renal tissue sections were then stained with 500 µl TUNEL reaction mixture from the TUNEL staining kit (Roche Diagnostics) for 60 min at 37°C in the dark, and TUNEL-positive cells were observed under an optical microscope (Olympus Corporation). The rate of apoptosis was calculated as follows: (number of TUNEL-positive cells/total number of cells) ×100%.