Immunofluorescence Staining of Chondrocytes

Immunofluorescence staining was conducted following standard protocols and our previous methods [19 (link)–21 (link)]. Chondrocytes that had undergone suitable treatment were fixed with 4% PFA for 30 minutes and permeabilized in 0.3% Triton X-100 at room temperature (RT) for 30 minutes. After incubating with 10% FBS for 1 hour at RT to block nonspecific binding, the samples were incubated overnight with primary antibodies against MYD88 (1:200, Abcam), collagen II (1:200, Abcam), MMP13 (1:200, Proteintech), NLRP3 (1:200, Affinity), GSDMD (1:100, Affinity), p-IκBα (1:200, Affinity), and p-p65 (1:200, CST) at 4° C. Next, the samples were incubated with secondary antibodies (1:300, Invitrogen). Finally, the fluorescence intensity of positive cells was observed under fluorescence microscope.