For immunohistochemistry, sections of the arteries were dewaxed and dehydrated as described previously. Immunohistochemical analysis was performed using Universal Two-step Immunohistochemistry Kit (PV-9000, zsbio, Beijing, China) according to the manufacturer’s instructions. Briefly, antigens were retrieved by trypsin, endogenous peroxidase was removed by 3% hydrogen peroxide, followed by 5% BSA blocking for 1 h. Sections were then incubated overnight at 4 °C with primary antibody for Runx2 (bs-1134R, 1:250, bioss), BMP2 (bs-10696R, 1:250, bioss), BMP4 (bs-1374R, 1:250, bioss), Crim1 (bs-21654R, 1:250, bioss). The next day, sections were incubated with reaction enhancer and secondary antibody, and positive areas were detected with DAB chromogenic solution. Finally, nuclei were stained with hematoxylin.
For immunofluorescence, frozen sections of aortas were incubated with the exosomal marker TSG101 rabbit antibody (bs-1365R, 1:200, bioss) and VSMC marker α-SMA mouse antibody (GB13044, 1:200, Servicebio). Subsequently, the binding primary antibodies was visualized using FITC/Cy3-conjugated secondary antibody. The nuclei were stained with DAPI. Sections were observed and photographed under a fluorescence microscope (Nikon Instruments Korea, Seoul, Korea).
For immunofluorescence, frozen sections of aortas were incubated with the exosomal marker TSG101 rabbit antibody (bs-1365R, 1:200, bioss) and VSMC marker α-SMA mouse antibody (GB13044, 1:200, Servicebio). Subsequently, the binding primary antibodies was visualized using FITC/Cy3-conjugated secondary antibody. The nuclei were stained with DAPI. Sections were observed and photographed under a fluorescence microscope (Nikon Instruments Korea, Seoul, Korea).