Immunofluorescence Staining of Chondrocytes

Condylar and disc chondrocytes were cultured in 24-well plates at a density of 20 000 cells per cm² overnight. Immunofluorescence staining of monolayer culture samples was performed as previously described.^{18 (link)} In brief, the monolayer culture samples were fixed in 4% paraformaldehyde (Sigma, St. Louis, MO, USA), permeabilized with 0.3% Triton X-100 (Sigma, St. Louis, MO, USA) and incubated overnight at 4 °C with primary antibodies for collagen VI, collagen IV and laminin. After the incubation, the cells were washed twice with 0.3% Triton X-100 in PBS and incubated with an Alexa Fluor 594 goat anti-rabbit secondary antibody (A11012; Life Technologies, Carlsbad, CA, USA). Nuclear staining was performed using 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies, Carlsbad, CA, USA) before imaging under an inverted fluorescence microscope (Olympus, Tokyo, Japan).