Bs 1374r

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).

Western blotting was performed to explore M2 polarization of macrophages and the level of skin fibrosis. The following antibodies were used in the western blotting assays: alpha-smooth muscle actin (α-SMA, 1:100, ab8211, Abcam); IL-10 (1:100, ab34843, Abcam); type I collagen (Col Ⅰ, 1:200, ab260043, Abcam); IL-6 (1:500, ab6672, Abcam); cytokeratin 17 (1:500, ab109725, Abcam); TNF-α (1:1000, ab6671, Abcam); arginase-1 (1:500/1:50, #93668, Cell Signaling Technology); CD206 (1:1000, #PA5-114310, Invitrogen); Smad1/5 (1:500, bs-2973R, Bioss Antibodies); phospho-Smad1/5 (1:1000, bs-3418R, Bioss Antibodies); BMP4 (1:500, bs-1374R, Bioss Antibodies); and GAPDH (1:10000, ab181602, Abcam).

The protein expression was determined by Western blotting as previously described [37 ]. 30 µg protein extracts were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (IPVH00010, PVDF, Millipore, Billerica, MA). After blocking with 5% non-fat milk for 1 h, the membrane was incubated overnight at 4 °C with primary antibody, including CD9 (ab92726, 1:1000, abcam), CD81 (ab109201, 1:1000, abcam), TSG101 (bs-1365R, 1:1000, bioss), Runx2 (ab23981, 1:2000, abcam), BMP2 (bs-10696R, 1:1000, bioss), GAPDH (10494-1-AP, 1:4000, proteintech), BMP4 (bs-1374R, 1:1000, bioss), Crim1 (bs-21654R, 1:1000, bioss), followed by incubation with the horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The immunoreactive bands were visualized by the enhanced chemiluminescence reagent (WBKLS0100, Millipore, Billerica, MA) and imaged by Amersham Imager 600 analyser (General Electric, USA).