Milli mark fluoropan neuronal marker

Neurospheres at passage 60 under growth conditions were washed with HBSS, fixed and treated with primary antibodies, anti-SOX2 (Santa Cruz Biotech, Santa Cruz, CA) and anti-nestin (Sigma–Aldrich). Neurospheres (60th passage) plated on poly l-lysine-coated 8-well Lab-Tek chambers (Thermo Scientific, Waltham, MA) in FGF-2/EGF-free medium were stimulated with 10 ng/ml PDGF-AA (Peprotech), 100 ng/ml BMP2 (Peprotech) or 5 μM forskolin (Sigma–Aldrich) for 5 days, fixed, and treated with primary antibodies, anti-DCX (Santa Cruz) for neuroblasts/neural precursors, Milli-Mark FluoroPan Neuronal Marker (Millipore, Billerica, MA) for neurons, anti-GFAP (Sigma–Aldrich) for astrocytes, anti-NG2 (Abcam, Cambridge, UK) for oligodendrocyte precursors, anti-O4 (Millipore) for oligodendrocytes, anti-MAP2 (Sigma–Aldrich) for neurons, anti-glutamate (Sigma–Aldrich) and/or anti-GABA (Sigma–Aldrich). In some experiments, frozen brain sections (30-μm thickness) were treated with primary antibodies, anti-SOX2, anti-BrdU (Abcam), anti-MAP2 and/or anti-GFAP. Each primary reaction was followed by reaction with an appropriate fluorescence-conjugated second antibody. Fluorescent signals were observed by a fluorescent microscopy (AXIO Imager A2, Carl Zeiss, Oberkochen, Germany) and a confocal laser scanning microscope (FV10i, Olympus, Tokyo, Japan).