Rat 401

Cells were fixed with 4% paraformaldehyde (PFA; Merck) at room temperature for 30 min and permeabilized with PBS containing 0.1% Triton X-100 for 15 min. Then, cells were blocked with 3% normal donkey serum (Sigma-Aldrich) for 1 h at room temperature and treated with primary antibody overnight at 4 °C. Primary antibodies were used as follows; rabbit anti-βIII-Tubulin (1:2000, PRB-435P; Biolegend), rat anti-MBP (1:500, ab7349; Abcam), rabbit anti-MAG (1:500, 9043; Cell Signaling Technology), mouse anti-GFAP (1:1000, GA-5, Sigma Aldrich), rat anti-GFAP (1:500, 2.2B10; Thermo Fisher Scientific), goat anti-PDGFRα (1:1000, AF1062; R&D Systems), rabbit anti-Ki67 (1:500, ab16667; Abcam), mouse anti-Olig2 (1:1000, 211F1.1; Millipore), rat anti-Nestin (1:1000, rat-401; Millipore). After three times wash, cells were incubated with secondary antibodies at room temperature for 1 h. Secondary antibodies were used as follows, Alexa 488-conjugated donkey anti-mouse IgG, Alexa 594-conjugated donkey anti-rat IgG (1:500; Thermo Fisher Scientific) and DAPI (1:2000; DOJINDO). After three times wash, cells were observed with IN Cell Analyzer 2000 (GE Healthcare) or confocal laser scanning microscope (FV3000, Olympus).