The articular cartilage tissues were fixed in 4% paraformaldehyde for 24 h. Then, the tissues were dehydrated with gradient ethanol, paraffin-embedded and sliced into 5-μm sections. After being dewaxed, the tissue sections were dehydrated by gradient ethanol, immersed in 3% H2O2 for 10 min, followed by high-pressure antigen retrieval for 90 s. Afterwards, the sections were blocked with 100 μL 5% bovine serum albumin (BSA) solution at 37 °C for 30 min. The sections were then incubated with 100 μL rabbit anti SDC1 (1:500, ab128936, Abcam,Cambridge, MA, USA) overnight at 4 °C. The next day,the sections were incubated with biotin-labeled secondary antibody goat anti-rabbit (HY90046, Shanghai Hengyuan Biotechnology Co., Ltd., Shanghai, China) at 37 °C for 30 min. Then, the sections were incubated with streptavidin-peroxidase (Beijing Zhongshan Biotechnology Co., Ltd., Beijing, China) at 37 °C for 30 min and colored by diaminobenzidine (DAB) (Bioss Biotech, Beijing, China) at room temperature. Finally, the sections were counterstained by hematoxylin for 5 min,differentiated by 1% hydrochloric acid alcohol for 4 s and blued under running water for 20 min. The SDC1 positive cells were analyzed using Image-Pro Plus image analysis software (Media Cybernetics, Silver Springs,MD, USA). The brownish-yellow colored cells were considered as positive cells (Kelkar et al. 2017). Five highpower fields (× 200) were randomly selected from each section, with 100 cells counted in each field. The percentage of positive cells = the number of positive cells/the number of total cells and the percentage of positive cells > 10% was regarded as positive (+), < 10% as negative (−).The experiment was repeated three times independently.
