Photomicrographs depicting immunofluorescence analysis of neurospheres labeled for intermediate filament proteins nestin (A), and the neuronal lineage marker, the neuronal-specific nuclear antigen, NeuN (B and lower magnification inset). Cell nuclei were counter-stained with DAPI (blue). Control neurosphere cultures are immuno-positive for nestin. However, cultures do not exhibit nuclear localization of NeuN, showing that neurosphere cultures were comprised of immature cells. (C) Differentiation following removal of the mitogen EGF, and dispersion of cells onto a laminin substrate, results in the upregulation and nuclear localization of NeuN. Photomicrograph represents a digitally merged immunofluorescence and phase contrast microscopic image showing NeuN immunofluorescence overlying nuclei (white arrows) of early differentiating neurons. Scale bars: 50 uM.