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.