Double-staining was done to confirm the phenotype and degree of differentiation. The pluripotent stem cell mark, OCT4 (goat, 1: 400, Santa Cruz Biotechnology), was stained to assess retention of pluripotency. Proliferating cells were stained with a monoclonal antibody against proliferating cellular nuclear antigen (PCNA, 1:500, Calbiochem, Gibbstown, NJ). Assessment of neural stem cell gene expression was done by the immunostaining of Sox2 (rabbit, 1:750, Millipore) or nestin (mouse, Developmental Studies Hybridoma Bank-University of Iowa, 1:400). Immunostaining for glial cells was performed with polyclonal antibody against the glial fibrillary acidic protein (GFAP, 1:400, DAKO, Carpinteria, CA). Controls included omission of the primary antibody, incubating with pre-immune serum, or preincubation of antigen to assure the specificity prior to the staining of NSC.
