2.8 Immunofluorescence cell staining
Cultured VSMCs were washed three times with PBS and fixed with 4%
paraformaldehyde for 15 min, followed by permeabilization with 0.1%
Triton-X-100 (Beyotime, China) for 30 min. Then cells were washed three
times with PBS and incubated with anti-phospho-Smad3 (anti-p-Smad3)
antibody (1:200 dilutions, ab52903, Abcam, England) overnight at 4°C,
followed by secondary goat anti-rabbit IgG-Cy3 antibody (1:200
dilutions, A0516, Beyotime, China) incubated for 1 h at room
temperature. Nuclear staining of the cells involved using
4’6-diamidino-2-phenylindole dihydrochloride (DAPI, Beyotime, China) for
5 min at room temperature. Images were captured by fluorescence
microscopy (OLUMPUS, Japan).