Immunofluorescence staining

Cultured CFs on sterile glass cover slips were washed briefly with cold PBS 3 times and fixed with 4% paraformaldehyde for 15 min. Then, the cell membrane was permeabilized by 0.4% Triton X-100 for 1 h and blocked by normal goat serum for 1 h, at 37°C. The cells were incubated with anti-α-SMA antibody (Abcam Inc., USA, ab7817, 1:100) and anti-vimentin antibody (Cell Signal Tech, #5741, 1:100) overnight at 4°C and subsequently incubated with a FITC-conjugated goat anti-mouse antibody for 1.5 h. The cells were then washed with PBS, and the nuclei were stained with DAPI (Roche Molecular Biochemicals) for 5 min at room temperature. Immunofluorescence was analyzed under a fluorescence microscope (Nikon 80i, Japan).