Gtx37732

Initially, the paraffin sections were removed using xylene after warming for 1 h in a wet box. Subsequently, enzymatic digestion with hyaluronidase (18240-36; Nacalai Tesque, Inc., Kyoto, Japan) was performed to expose the antigens in the specimens. Additionally, to minimize non-specific reactions, the specimens were blocked using 3% bovine serum albumin. Then, the specimens were subjected to incubation with the following primary antibodies at 4 °C: anti-ki67 (× 200, M7240, Abcam, Cambridge, United Kingdom), anti-TNAP (× 300, ab65834, Genetex), anti-ENPP1 (× 300, rabbit, Genetex), anti-Col2 (× 300, MA5-12789, Thermo Fisher, Tokyo, Japan), and anti-Col10 (× 300, GTX37732, Genetex). Finally, the specimens were further incubated with secondary antibodies for 30 min at 25 °C and then mounted with a DAPI-containing mountant for fluorescence observation (Abcam). The histological analysis, specifically the counting of Ki67-positive cells, was performed on samples from three rats, with all positive cells counted through visual inspection. Furthermore, standardized exposure times of 250 ms for Ki67 and 3 ms for DAPI were applied, and the enumeration of positive cells was performed visually. For light observations, TNAP and ENPP1 were combined with diaminobenzidine.

The anti-collagen II (Ab79127) and anti-collagen X (GTX37732 from Genetex) were used after treatment of fixed cells (15 min in 4% paraformaldehyde) with hyaluronidase 5 mg/ml for 30 min at 37°C. MMP13 was detected with anti-MMP13 (Ab39012 from Abcam). Quantification of immunohistochemical staining was done by image binarization and normalization of positive pixels over total cells. At least three independent fields were quantified per condition. Murine and human XOR expression was evaluated using an anti-XOR rabbit polyclonal antibody (5 μg/ml final concentration, sc-20991, Santa Cruz Biotechnology Inc., Dallas, TX, United States) on knee cartilage paraffin section. Semi-quantitative scoring was performed by two independent observers based on the following scale in three different fields per sample: 0 (no XOR expression); 1(up to 20% positivity); 2 (between 20%-40% positivity); 3 (between 40%-60% positivity); and 4 (above 60% positivity).