Double-staining was done to confirm the phenotype and degree of neuronal differentiation. The pluripotent stem cell marker, OCT4 (goat, 1:400, Santa Cruz Biotechnology), was stained to assess retention of pluripotency. Neurons were identified by monoclonal antibody against microtubule associated protein a, b, and c (MAP2, Sigma, 1:500; detecting immature and mature neurons). DNA methylation was investigated using antibodies against 5-methyl-cytosine (5-MeC, goat-anti-5-MeC, 1:250, GeneTex, San Antonio, TX). Changes in expression of the enzyme responsible for DNA methylation, DNA methyltransferase (DNMT), were immunostained using polyclonal antibodies against DNMT1 (de novo DNA methylation; made in goat, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA). Endogenous peroxide was quenched with 3% H2O2 and 1% Triton X-100 was applied for 30 minutes to permeabilize the cell membranes. For 5-MeC staining, cells were incubated with 2N HCl. Non-specific binding was blocked using 4% normal serum from the species used to make the secondary antibody, plus 0.1% Tx-100 in PBS. Primary antibodies (detailed above) were incubated overnight at room temperature in the blocking buffer corresponding to the species of the secondary antibody. For immunofluorescent staining, the NSCs
