Immunohistochemical Evaluation of Cartilage Markers

The paraffin-embedded tissue sections were deparaffinized and rehydrated following standard procedures. Sections were incubated with 3% H₂O₂ to block endogenous peroxidase activity and antigen retrieval was performed in citrated buffer at 110 ℃, for 5 min in a pressure cooker. After the citrated buffer reached room temperature, the sections were removed and incubated overnight with the primary antibodies COL2A1 (1:200, bioss, bs-10589R) and SOX9 (1:1000, Abcam, Cat# ab185966) at 4 ℃, followed by incubation with an HRP conjugated secondary antibody (Beyotime Institute of Biotechnology, Inc., Nantong, China) for 2 h at room temperature. Peroxidase binding for both COL2A1 and SOX9 was visualized using diaminobenzidine. Then, the nuclei were counterstained with hematoxylin, while the slides were dehydrated, mounted, and analyzed with a light microscope. For the quantitative analysis, all positively stained cells, including those in the femoral condyle and tibial plateau area, on the articular surface per specimen were counted, and the percentage of positive cells was calculated using Image-Pro Plus 6.0.