Ix71 f22ph

Serial thoracic aorta sections (4 μm) were deparaffinized with xylene, rehydrated in graded ethanol, and pretreated with 0.01 M citric acid buffer (pH=6) for 20 min in a thermostatic water bath (99 °C). All materials were pre-incubated with 3 % hydrogen peroxide in phosphate-buffered saline (PBS) for 5 min to ensure endogenous peroxidase inactivation. The sections were then incubated with 0.3 % TritonX-100 for 15 min, washed 3 times in 0.01 M PBS for 5 min each, and blocked with 5 % normal goat serum in PBS for 20 min. Next, the samples were incubated with anti-α smooth muscle actin (ab5694, Abcam, USA; 1:200), anti-Osteopontin (ab63856, Abcam, USA; 1:100), or anti-Calponin 1 (sc58707, Santa Cruz Biotechnology, USA; 1:200) primary antibodies overnight at 4 °C. Following incubation, the sections were incubated with biotinylated goat anti-rabbit IgG (SA00001-2, Proteintech Group, USA) or anti-mouse IgG (SA00001-1, Proteintech Group, USA) secondary antibodies at a concentration of 1:200 in PBS for 1 h. The sections were then washed 3 times with PBS, visualized with 0.05 % 3-3'diaminobenzidine (DAB) solution, counterstained with Harris hematoxylin, and imaged on an inverted microscope (IX71-F22PH; Olympus, Tokyo, Japan). Control sections were treated with only secondary antibody.

For morphological examination, thoracic aortas were carefully dissected from the connective tissue to avoid mechanical stretching and perfused with 4 % paraformaldehyde for 12 h. The vessels were then rinsed with phosphate buffered solution, dehydrated with ethanol, embedded in paraffin, and cut into 4 μm sections. These tissue sections were stained with hematoxylin and eosin (HE), and images were obtained using an inverted microscope (IX71-F22PH; Olympus, Tokyo, Japan).