The self-renewal and differentiation markers of the NPCs were also assessed by immunofluorescence34 (link). For immunofluorescence staining analysis cells were incubated with the primary antibody Nestin (1:400; MAB353, Millipore), Tuj1 (1:500,05-549, Upstate), Sox2 (1:100; 481400, Life technologies), Map2(1:400; M1406, Sigma), Vimentin (1:100; V6630, Sigma), GFAP (1:500; MAB360, millipore) overnight at 4 °C. The secondary antibodies are anti-mouse IgG FITC antibody (1:200, St. Louis, MO, USA) and anti-rabbit IgG FITC antibody (1:1000, St. Louis, MO, USA) diluted in blocking buffer. Nuclei are counter-stained with Hochest 33342 (1:500; 94403, St. Louis, MO, USA). The fluorescent images of 2-D cultured cells were visualized on a Zeiss 200 inverted fluorescent microscope (Carl Zeiss, Jena, Germany). The number of immunostained cells was counted in each of three random fields per well and the fluorescence images were selected randomly. The quantification of the immunofluorescence signal was performed by Image-Pro Plus software (Media Cybernetics, Bethesda, MD). The fluorescent images of 3-D cultured cells were taken with a Leica TCS SP5 scanning laser confocal fluorescence microscope (Leica Microsystems, Inc., Germany).
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