Fluoview software version 3

After post-fixation, brains were washed in 0.1 M PB and cut into serial 10-μm thick coronal sections using a cryostat (Leica, CM 1900). One series (five to eight sections) from each animal was used in each immunostaining. Sections were incubated in blocking solution for 1 h at room temperature, followed by overnight incubation at 4°C with primary antibodies (see Table 1). Then, sections were washed and incubated with the appropriate secondary antibodies conjugated with either biotin or fluorophores. After the secondary biotinylated antibody, sections were incubated with ABC Elite complex (Vector, Burlingame, CA) and treated with diaminobenzidine (DAB, 0.05%; Sigma–Aldrich). Measurement of BrdU incorporation during DNA synthesis was carried out in coronal sections by quantification of BrdU+ cells within the SVZ, under an Eclipse E200 light microscope (Nikon, Tokyo, Japan), and were expressed as cells/mm. Fluorescence samples were examined under an Olympus IX81 confocal microscope and imaged using the Olympus Fluoview software version 3.1(Center Valley, PA).