TUNEL assay

Articular cartilage tissue sections were fixed in 4% paraformaldehyde for 24 h at room temperature and embedded in paraffin. Then, 4 µm-thick paraffin sections were deparaffinized using 100% xylene, rehydrated for 5 min twice at room temperature and washed with H₂O. This was followed by rehydrated with ethanol at graded concentrations (100% 5 min, 100% 3 min, 95% 3 min, 85% 3 min, 70% 3 min, 50% 3 min) at room temperature. Then, the tissues sections were treated with 100 µl proteinase K [20 µg/ml; Roche Diagnostics (Shanghai) Co., Ltd.)] for 20 min at room temperature, and washed 1X PBS. Subsequently, chondrocyte apoptosis in the articular cartilage was measured using a In situ Cell Death Detection kit (cat. no. 11684817910; Roche Diagnostics Co., Ltd.), according to the manufacturer's protocols. Cells with brown nuclei were deemed TUNEL-positive and were counted by a microscope using three fields of view/section. A light microscope (Olympus Corporation; model BX51) was used to capture the images (magnification, ×200).