Immunostaining procedures followed a routine procedure as previously established for stem cell cultures in our laboratory (8,26). In brief, endogenous peroxide was quenched with 3% H2O2 and 0.3% 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 were washed with PBS and incubated with an Alexa 488 or 635 fluorophor conjugated secondary antibody (against the primary antibody species) at room temperature for 90 minutes and counterstained with 4',6-diamidino-2-phenylindole (DAPI- A-T-specific DNA stain at 350nm wavelength, Invitrogen). Staining of undifferentiated NSCs was done in a 1.5mL Eppendorf centrifuge tube following the procedures above.
