Bs 10589r

Following micro-CT, the limbs were placed in 4% paraformaldehyde solution for 2 days and then decalcified for 12 days in 20% ethylene diamine tetra acetic acid(EDTA) in PBS (PH 7.0), processed, and embedded in paraffin wax, and 5μm coronal sections were obtained following the OARSI [44 (link)]. Serial sections were harvested every 75μm to encompass all weight-bearing areas of the knee joint. The sections were stained with 0.1% Safranin O/0.02% fast green to detect proteoglycans and glycosaminoglycans. Immunohistochemistry staining for Collagen II (Col2a1) (1:100, bs-10589R; Bioss, Beijing, China), Aggrecan (Acan) (1:200, bs-11655R; Bioss), MMP3(1:50, Ab52915; Abcam, Cambridge, UK), MMP13 (1:200, Ab39012; Abcam), IL6 (1:100, ab290735; Abcam) and TNFα(1:1000, ab307164; Abcam) were conducted on paraffin sections following appropriate antigen retrieval methods. The sections were imaged via an Axio Scope Light Microscope (Zeiss). The tibial plateau quadrants of the knee joint were scored by two independent blinded observers according the mouse recommendations of OARSI [44 (link)]. OA severity is expressed by the mean maximum score.

Immunohistochemical (IHC) was used to measure the distribution of NLRP3, Caspase‐1, MMP‐3, and Collagen II in vertebral disc tissue. After dehydration, the sections were placed in 0.01 M citrate buffer (pH 6.0) and kept at a high temperature for 20 min. After the sections were cooled and washed, 1% periodic acid was used for 10 min to eliminate endogenous enzymes. Next, NLRP3 primary antibody (PA5‐79740, 1:400, Cell Signaling Technology, Danvers, MA, USA), Caspase‐1 primary antibody (22915‐1‐AP, 1:100, Proteintech, Chicago, IL, USA), MMP‐3 primary antibody (17873‐1‐AP, 1:100, Chicago, IL, Proteintech, USA), and Collagen II primary antibody (bs‐10589R, 1:200, Bioss, Beijing, China) were incubated. The secondary antibodies were then added for incubation. Subsequently, the sections were cultured with 3,3′‐diaminobenzidine (DAB), hematoxylin, and alcohol dehydration. Finally, the sections were blocked with a neutral resin and examined under a microscope (BA410T; Motic, Hongkong, China).