Neural progenitor cells derived from control dams were expanded as adherent monolayer cultures under either proliferating or differentiating conditions and were prepared for immunohistochemical analysis and imaging. Cells were transferred to poly-D-lysine coated coverslips after the second passage and grown in complete medium with growth factors EGF and FGF-2. After 2 days, cells were immunostained with the neural stem cell marker Nestin (1:1000; BD Biosciences) and counterstained with DAPI (0.5 μg/mL; Sigma) (Roitbak et al., 2011). To determine appropriate culture of neurons, cells were transferred to poly-D-lysine coated plates after the second passage and grown in complete medium without growth factors. After differentiation for 7 days without growth factors, cells were immunostained for astrocytes with GFAP (1:1000; Sigma-Aldrich) and neuroblasts and immature neurons were immunostained with doublecortin (1:1500; Cell Signaling) and counterstained with DAPI (0.5 μg/mL; Sigma). Secondary antibodies used for analysis included Alexa Fluor 647 (1:250; Invitrogen; A-21236) and Cy3 (1:500; Invitrogen; A10520). Images for publication were acquired using a Zeiss LSM-510 META confocal microscope equipped with a laser diode, one argon laser, and two HeNe lasers. Maximum intensity projections of Z stacks were acquired with a 20X objective.
