Immunofluorescence Assay for α-SMA Expression

KFs were seeded into 15 mm glass bottom cell culture dishes at a density of 4.0 × 10⁴ cells per well with different drug treatments for 48 h. After being washed with PBS, the cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 (Solarbio, China) for 15 min, blocked with 5% BSA at room temperature for 1 h, and incubated with anti-α-SMA antibody (Proteintech, US) overnight at 4°C. On the next day, the cells were incubated with CoraLite594-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (Proteintech, US) for 1 h at room temperature in the dark. DAPI (Sigma-Aldrich, US) was used for nuclear staining. Images were obtained using a TCS SPE confocal microscope (Leica, GER). Images were analysed by IPP 6.0 [18 (link)].