Immunofluorescence Assay for Collagen II and MMP-13

Immunofluorescence experiments were carious out according to previously described [49 (link)]. Chondrocytes from various treatment groups were washed twice in PBS before being used to make cell smears. The cells were fixed for 30 minutes at room temperature with 4 percent paraformaldehyde, washed twice with PBS, and then treated for 15 minutes at room temperature with 0.1% Triton-X 100 to render them permeable. After washing the permeate, Collagen II (1:200 dilution, Abcam, UK) and MMP-13 antibody (1:200 dilution, Abcam, UK) were added and the reaction was incubated overnight at 4°C. After washing out the first antibody, a fluorescently labeled secondary antibody (1:200, Abcam, UK) was added and incubated at room temperature for 1 hour before being washed three times with PBS. After adding DAPI staining and washing out the staining solution the cells were observed under the fluorescence microscope (Olympus DP80, Japan).