Immunofluorescence

Briefly, cells in 24-well plates were rinsed twice with phosphate-buffered saline (PBS), fixed for 15 min with 4% paraformaldehyde, permeabilized for 10 min with 0.2% Triton X-100, and then blocked for 30 min with normal goat serum (Beijing Zhongshan Jinqiao Biotechnology, Beijing, China). Subsequently, cells were incubated overnight with primary antibodies at 4ºC. The following day, cells were incubated with Alexa Fluor® 594 goat anti-rabbit immunoglobulin G (IgG) secondary antibody (Thermo Fisher Scientific) for 30 min at 37ºC. After washing three times with PBS, nuclei were stained for 5 min with 4',6-diamidino-2-phenylindole (DAPI; Invitrogen; Thermo Fisher Scientific). Images were acquired with an inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan). Primary antibodies were as follows: GFAP (1:1,000; Abcam) and Ki-67 (1:250; Abcam).