2.12. Immunofluorescence analysis of chondrocytes

After washing with PBS and being fixed with 4% paraformaldehyde for 20 min at room temperature, the cells were permeabilized with 0.5% Triton X‐100 for 30 min and incubated in nonspecific binding blocking solution (5% BSA) for 30 min at room temperature. Rabbit polyclonal anti‐NF‐κB p65 antibody (AB21014, 1:50; Absci) was added to the cells overnight at 4°C followed by staining with Alexa Fluor 488 conjugated anti‐rabbit antibody for 60 min at room temperature in darkness. The cytoskeleton was stained with phalloidine for 60 min at 37°C. Nuclei were counterstained with 4,6‐diamidino‐2‐phenylindole for 2 min. After washing, the cells adhered to Bioflex membranes were mounted in PBS with 20% glycerol. The chondrocytes were visualized with a confocal microscope (Olympus, Tokyo, Japan).