Sm α actin

Immunofluorescence of PASMC was conducted as previously described [19 (link)]. PASMC was seeded in a six-well plate and grown to 50%–60% confluence. After removal of cell culture media, cells were washed twice by PBS and fixed with 4% polyformaldehyde. PASMC was then incubated with 0.5% triton X-100 T for membrane breaking. After blocking, the cell was incubated with primary antibodies against SM α-actin and Ki-67 (1:1000; Cell Signaling Technology) overnight in dark at 4°C. PASMC was then rinsed by PBS and incubated with relative secondary antibodies (Alexa Fluor 594 F(ab’)-conjugated goat anti-mouse and Alexa Fluor 488-conjugated goat anti-rabbit (1:3000; Molecular Probes Inc., Eugene, Oregon, USA)) in dark for 1 h, respectively. The nuclei were stained with DAPI (5 mg/ml; VECTOR Labs, Burlingame, California, USA) for 5 s in the room temperature. Images were captured with immunofluorescent microscopy (Leica MPS 60, Germany).

IF staining was conducted as described previously [46 (link)]. Cell plates were firstly washed by PBS for three times, followed by fixation with 4% paraformaldehyde for 20 min and then blocked in 1% blocking solution. The VSMC was then incubated overnight with primary antibodies against SM α-actin and Ki-67 (1: 1000; Cell Signaling Technology) in dark at 4°C. After washed with PBS, VSMC was then incubated with Alexa Fluor 594F(ab’)-conjugated goat anti-mouse and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibodies (1: 2500; Molecular Probes Inc., Eugene, OR), respectively, for 1 h in dark. Subsequently, VSMC was incubated with DAPI (5mg/ml; VECTOR Labs, Burlingame, CA) for 5 s in the room temperature. Images were acquired by using an immunofluorescent microscopy (Leica MPS 60; Wetzlar, HD). The fluorescence intensity was analyzed by using Image J software (Bethesda, MA).