Immunofluorescence for Cell Characterization
Identification of the isolated AMDC was performed by immunofluorescence cell staining with AM patients. In brief, cells were seeded on climbing piece and fixed with 4% paraformaldehyde universal tissue fixative for 10 mins at Room temperature, followed by 0.5% Triton X-100 (Solarbio, T8200, Beijing, China) for 10 min, Normal Goat Serum, 10% (Bioss, C01-03001, Beijing, China) for 30 min. Thereafter, with primary antibody with the whole night at 4℃, Rabbit Anti-Vimentin antibody (VIM, 1:100, bioss, bs-0756R, Beijing, China), Rabbit Anti-CK7 antibody (1:100, bioss, bs-1610R, Beijing, China), Rabbit Anti-Von Willebrand Factor antibody (VWF, 1:100, bioss, bs-10048R, Beijing, China), Rabbit Anti-Ecadherin antibody(1:100, bioss, bs-10009R, Beijing, China), Rabbit Anti-alpha smooth muscle Actin antibody(SMA, 1:100, bioss, bs-10196R, Beijing, China), PBS (Servicebio, G4202, Wuhan, China), respectively. Followed by incubated with the secondary antibody of goat anti-rabbit IgG (H +L) (1:400; YEASEN, Shanghai, China) at 1h on second day, DAPI (Solarbio, C0065, Beijing, China)5 min. Before each next step, washing with PBS 3 times for 5 min.