Alexa fluor 594 conjugated antibody

Primary chondrocytes were cultured in glass slides with the complete culture medium supplemented with 10 ng/mL IL-1β for 24 h and then incubated with AT for 48 h. Following this, the cells were washed with 1× PBS and fixed in 4% paraformaldehyde for 20 min at room temperature. Fixed cells were permeabilized with 0.1% Triton X-100 for 10 min and blocked with 5% BSA for 1 h. Subsequently, primary antibodies against MMP-3 (Abcam, 1:100), beclin-1 (Abcam, 1:100), and TNFSF11 (Abcam, 1:100) with 3% BSA were used to treat cells at 4 °C overnight. Following this, the cells were washed with 1× PBS three times, and incubated with secondary Alexa Fluor 594-conjugated antibody (Abcam,1:200) and Molecular Probes Alexa Fluor 488 phalloidin (Sigma–Aldrich) for 1 h at room temperature. The cell nuclei were then stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma–Aldrich) for 5 min, coverslips were mounted on glass slides, and cells were imaged using a fluorescence microscope.