Apochromat 63 oil immersion objective

Freshly isolated VSMCs were fixed with 4% paraformaldehyde (Sigma‐Aldrich) for 10 min, washed with PBS, and permeabilized with PBS containing 0.1% Triton X‐100 for 20 min at room temperature. Cells were incubated with PBS containing 1% BSA for 1 h at room temperature and then were incubated with primary antibodies in PBS containing 1% BSA overnight at 4°C. In control experiments, cells were incubated without the primary antibody. The cells were washed and incubated with secondary antibodies conjugated to a fluorescence probe. Unbound secondary antibodies were removed by washing with PBS, and nuclei were labelled with 4′,6‐diamidino‐2‐phenylindole (DAPI) mounting medium (Sigma‐Aldrich). Cells were imaged using an LSM 510 laser scanning confocal microscope (Carl Zeiss). The excitation beam was produced by an argon (488 nm) or helium/neon laser (543 nm and 633 nm) and delivered to the specimen via an Apochromat 63× oil‐immersion objective (numerical aperture, 1.4) (Carl Zeiss). Emitted fluorescence was captured using LSM 510 software (release 3.2; Carl Zeiss). Two‐dimensional images cut horizontally through approximately the middle of the cells were captured (1024 × 1024 pixels). Raw confocal imaging data were processed and analysed using LSM 510 software. Final images were produced using PowerPoint (Microsoft Inc.).