Ab34712

Following the completion of micro‐CT scanning, the specimens were preserved in a 70% ethanol solution. Subsequently, they underwent decalcification in a 14% EDTA solution for a period of 14 days, culminating in paraffin embedding. Afterwards, sections with a thickness of 4 μm from the medial compartment of the joints were prepared for Alcian Blue Haematoxylin/Orange G and Toluidine Blue staining, aiming to assess the overall structural changes in the cartilage. Histomorphometric analysis was conducted using OsteoMeasure software (Decatur, GA). Three blinded observers scored the cartilage structure degeneration based on the guidelines provided by the Osteoarthritis Research Society International (OARSI). The expression of Collagen TypeII (Col2), matrix metalloproteinase 13 (MMP13) was observed by immunohistochemistry. Anti‐Col2 (Abcam, ab34712, 1:200), anti‐Aggrecan (Bioss, bs‐11655R, 1:200) and anti‐MMP13 (Abcam, ab39012, 1:100) were utilized in this study.

For IHC, sections were toasted at 60°C for 4 hours before deparaffinization and dehydration. Slices were then soaked in sodium citrate solution and heated at 60°C for 4 hours to make antigen retrieval. Subsequently, sections were incubated with primary Abs at 4°C overnight and treated for secondary Abs for 20 minutes the next day. The positive signal was visualized with DAB reagents (ZSGB-BIO) and slices were counterstained with hematoxylin. For immunofluorescence (IF), samples were incubated with a fluorescent secondary Ab for 40 minutes at room temperature and DAPI was used for nuclear staining. The primary Abs employed in the current study include Col2a1 (Abcam, catalog ab34712; 1:500, IF), PPM1A (Bioss, catalog bs-4162R; 1:300, IHC; and Abcam, catalog ab14824; 1:100, IF), MMP-13 (Abcam, catalog ab39012; 1:300, IHC), Adamts-5 (Bioss, catalog bs-3573R; 1:200, IHC), and p-SMAD2 (Ser465 and Ser467) (Thermo Fisher Scientific, catalog 44-244G; 1:300, IHC/IF). Quantitative analysis was performed using ImageJ software.