α sma

Human aortic samples were fixed in 10% formalin, embedded in paraffin, and sectioned at 5 μm intervals. Immunohistochemical staining was performed using established methods [17 (link)]. To remove the paraffin, sections were treated with xylene and rehydrated. Then, they were incubated with 3% H₂O₂ for 10 min at room temperature and washed 3 times with phosphate-buffered saline (PBS). After blocking with serum for 30 min, the sections were incubated with primary antibodies against YAP (1 : 1000 dilution, Cell Signaling), α- smooth muscle actin (α-SMA, 1 : 500 dilution, Sigma), Bcl-2 (1 : 1000 dilution, Cell Signaling), and cleaved caspase-3 (1 : 300 dilution, Cell Signaling), followed by incubation with the ChemMate EnVision System (Dako). ImageProPlus 3.0 (ECIPSE80i/90i) was used to capture the images and analyze the results. For cryostat sections, human and mouse aortic samples were fixed in 4% paraformaldehyde, embedded in optimum cutting temperature (OCT) compound, frozen in liquid nitrogen, and sectioned at 5 μm intervals. DeadEnd Fluorometric TUNEL (Promega) was used to detect the apoptotic cells. Apoptotic VSMCs were detected by TUNEL and α-SMA double staining before confocal fluorescence microscopy analysis (Leica Microsystems).