Immunofluorescence Staining of 3D-Cultured Neural Stem Cells

The differentiation status of the NSCs was also assessed by immunofluorescence staining, in accordance with a slightly modified version of procedures in previously published papers (Cui et al., 2016 (link)). For immunofluorescence staining analysis, cells were incubated with the primary antibodies Tuj1 (1:500,05-549, Upstate), Map-2 (1:400; M1406, Sigma), and GFAP (1:200; MAB360, Millipore) overnight at 4°C. The secondary antibodies were anti-mouse IgG FITC antibody (1:200; 31547, Invitrogen) and anti-rabbit IgG FITC antibody (1:1000; 31635, Invitrogen) diluted in blocking buffer. Nuclei are counter-stained with Hochest 33342 (1:500; Sigma). The fluorescent images of 3D-cultured NSCs were visualized on a Leica TCS SP5 scanning laser confocal fluorescence microscope (Leica Microsystems). The number of immunostained cells was counted in each of three random fields per well and the fluorescence images were selected randomly. Quantification of the immunofluorescence signal was performed using Image-Pro Plus software (Media Cybernetics, Bethesda, MD, United States).