The undifferentiated and differentiating neurospheres were defined as follows. The undifferentiated neurospheres (up to 100+ in 0.5-1mL culture media) were obtained from the above culture medium that was deprived of EGF+bFGF for 2 days. The neurospheres for differentiation experiments were withdrawn from EGF+bFGF supplementation and equilibrated for 5-10 minutes in Neurobasal media (no supplements) before being subplated into 16-well chamber slides (Nunc, Rochester, NY) coated with poly-D-lysine (20mg/mL, Sigma, St. Louis, MO) and laminin (1mg/mL, Sigma). No more than 3 neurospheres were placed per well. Neurospheres were allowed to differentiate for 4 days in differentiation media consisting of Neurobasal media supplemented with 10% fetal bovine serum, 1.2% B27, and 1.2% penicillin-streptomycin. All cells were fixed in freshly prepared fixative [4% paraformaldehyde (PFA) in phosphate buffer].
