α sma

Third‐order mesenteric arteries were isolated and cleared of adipose and connective tissues, followed by immediately fixed in 10% neutral‐buffered formalin solution. Embedded in paraffin, vessel rings were serially sectioned at 6 μm. According to the standard immunohistochemical staining procedures,29 Gpr41 and Olr59 were measured in the vessel sections using primary antibody (Gpr41, 1:100, Santa Cruz, USA; Olr59, 1:100, Sigma, USA). Nuclei were visualized with haematoxylin. Negative controls were performed by omission of the primary antibodies.
Vascular smooth muscle cells grown on coverslips were washed by PBS and fixed in 10% neutral‐buffered formalin solution. After incubation with PBST (PBS with 0.5% TritonX‐100) for 15 minutes, slides were blocked for 1 hour. Then, primary antibody of α‐SMA (Beyotime) and TRITC‐conjugated secondary antibody (Sangon) were applied. Nuclei were counterstained by DAPI (Beyotime). Negative controls were performed by omission of the primary antibodies.

Double IF was carried on the formalin-fixed, paraffin-embedded slides from human normal salivary tissues to examine PTEN (Zymed, San Francisco, CA, USA, 1:100, 18–0256) distribution compared with α-SMA (Beyotime Institute of Biotechnology, Jiangsu, China, 1:500, AA132), p63 (Maixin Biotech, Fujian China, ready-to-use, MAB0365), and Cytokeratin 5 (Thermo Scientific, San Jose, CA, 1:200, MS-1896) using the protocol we described previously [30 (link)]. Double IF labeling was achieved by mixing Alexa Fluor488-conjugated goat anti-rabbit IgG and Fluor594-conjugated goat anti-mouse IgG (Molecular Probes, Invitrogen, Carlsbad, CA, 1:500). Nuclei were labeled with 4, 6′-diamidino-2-phenylindole (DAPI, Beyotime Biotech, Jiangsu, China, 1:500). Images were collected with a BX43 fluorescence microscope (OLYMPUS) using cellSens software.