Sc 1851

Immunofluorescence staining was performed as previously described.^{18 (link)} In brief, HCASMCs were seeded on sterile coverslips that had previously been coated with 200 μL of 0.3% gelatin at room temperature for 30 minutes. After the cell confluence reached 90%–100%, the cells were preincubated with or without hesperetin for 30 minutes and then treated with SPC for 10 minutes. Next, the HCASMCs were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS for 2 minutes, blocked in NanoBio blocker solution (NanoBio Tech Co, Ltd, NY), and diluted in PBS for 60 minutes at room temperature. After that, the HCASMCs were stained with primary anti-ROCK (dilution time: 1:100; sc-1851, Santa Cruz, Dallas, TX) and anti-Fyn (dilution time: 1:100; 610164, BD Biosciences, NY) antibodies at 4°C overnight, followed by staining with Alexa Fluor 488-conjugated donkey antigoat IgG (A32814; Thermo Fisher Scientific, Waltham, MA) and Alexa Fluor 546-conjugated goat antimouse IgG (A11003; Thermo Fisher Scientific) secondary antibodies. Before observation, the cells were mounted with PermaFluor aqueous mounting medium (Thermo Fisher Scientific). The field of view was randomly selected to observe stained cells under an all-in-one fluorescence microscope BZ-9000 (Keyence, Osaka, Japan). Fluorescence intensity profile analysis was performed using Image J software.