Fluorescence detection system

β-Actin and CD63 expression were confirmed with western blot analysis; 2 mg/ml of hUC-MSC-EVs was separated by using 12% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred onto the polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membrane was blocked with 2% BSA for 1 h at room temperature and incubated with primary antibodies, rabbit monoclonal antibody CD63 (Abcam, Cat. No. ab134045) 1:2000 dilution, and rabbit monoclonal antibody β-Actin (Cell Signaling Technologies, Cat. No. 4970S) 1:5000 dilution overnight at 4 °C. The membrane was then washed with PBST and incubated with fluorescence secondary antibody goat polyclonal anti-rabbit IgG (Thermo Fisher Scientific, Cat. No. A16097) 1:10,000 dilution for 1 h at room temperature. The secondary antibodies were washed with PBST and developed using a fluorescence detection system (Licor).