Indirect immunofluorescence detection of antigens was carried out in hNPCs and differentiated neurons cultured on poly-ornithine/laminin-coated 96-well plates (Costar, Cambridge, MA) or similarly coated 24-well plates with glass coverslips. Cells were first washed twice with PBS and fixed for 30 minutes at room temperature with 4% PFA in PBS. After fixation, cells were washed twice with PBS, permeabilized with PBS-T (PBS and 0.25% Triton X-100) for 20 min, blocked in blocking solution (5% normal serum in PBS-T) for 30 min, and finally incubated overnight at 4°C with the first primary antibody in blocking solution. Next day, cells were washed with PBS and incubated for two hours at room temperature in the appropriate fluorophore-conjugated secondary antibody solution (Alexa 488- or 594-conjugated secondary antibody [Molecular Probes, Invitrogen] in blocking solution). After washes with PBS, cells were incubated again overnight in primary antibody solution for the second antigen and the procedure for conjugation of the fluorophore-conjugated secondary antibody was repeated as above. Finally, cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) and washed twice before imaging using a fully automated epifluorescence microscope equipped with
