Ki 67 primary antibodies

Ki-67 immunofluorescence staining was performed using the labeled streptavidin–biotin method to evaluate the proliferation of PAECs. After being washed three times with phosphate buffer saline (PBS), the PAECs were sequentially fixed in 4% paraformaldehyde at 4°C for 20 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and blocked with 3% bovine serum albumin in PBS for 1 h at room temperature. The cells were then incubated with the Ki-67 primary antibodies (1:500 dilution, Abcam, Cambridge, MA, United States) at 4°C overnight, and then stained with goat anti-rabbit IgG antibody (1:500, Thermo Fisher Scientific, Alexa Fluor 488) at 37°C for 1 h. Finally, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:500, Abcam). The Ki-67 immunostaining of PAECs were detected by a fluorescence microscope (Zeiss, Heidenheim, Germany). The percentage of Ki-67-positive cells was indicated as the proliferation rate of PAECs. Measurements were repeated three times.