Vascular smooth muscle cells were seeded on 13‐mm‐diameter cover slides. Then, the VSMCs were fixed using cold paraformaldehyde (4%) at 4°C for 20 minutes, followed by permeabilization with cold methanol (−20°C) for 5 minutes. Subsequently, the cells were incubated with a mouse anti‐smooth‐muscle (SM) α‐actin antibody (Sigma‐Aldrich, St. Louis, MO), calponin antibody (Abcam, UK) and MYH11 antibody (Abcam) at 4°C overnight. Next, the cells were incubated with an Alexa Fluor® 594 donkey antimouse IgG (H + L) secondary antibody (Invitrogen) at room temperature for 1 hour. Finally, the cells were visualized using a confocal microscope (Zeiss LSM 510; Germany).

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