The cells were fixed in PBS containing 4% paraformaldehyde for 10 min at room temperature. Thereafter, the cells were incubated with the blocking buffer (10% goat serum in PBS plus 0.05% Tween 20: PBS-T) for 1 h at room temperature. The primary antibodies were applied overnight at 4 °C. Detection was by the following primary polyclonal mouse antibodies; SOX2 (Abcam, Cambridge, MA, USA; dilution 1/200), NeuN (Abcam; dilution 1/500), βIII-tubulin (Merck Millipore; dilution 1/500), MAP2 (Sigma-Aldrich; dilution 1/1000), GFAP (Merck Millipore; dilution 1/750), S100B (Sigma-Aldrich; dilution 1/100), OLIG2 (Merck Millipore; dilution 1/200) and SOX10 (R&D Systems; dilution 1/100). After three washes in PBS-T, a secondary antibody (Alexa Fluor 488-labeled goat anti-mouse IgG, Life Technologies; dilution 1/1000) was applied for 1 h at room temperature. The cells were counterstained with DAPI (4′,6-diamidino-2-phenylindole) to highlight the nuclei. After washing in PBS-T, the cells were mounted in PermaFluor Aqueous Mounting Medium (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescent signals were detected using a confocal laser-scanning microscope FV1000 (Olympus, Tokyo, Japan). We counted cells positive for both βIII-tubulin and DAPI signals as neurons. These
