Sp8 white light laser scanning confocal microscope

For quantifications of apical surfaces of B1 cells across different ages, V-SVZ whole-mounts from P21, P60, P180 or P360 hGFAP::GFP mice (n= 3, 6, 7, and 3 mice respectively) were immunostained for GFP, beta-Catenin and gamma-Tubulin (Mirzadeh et al., 2008 (link)). Images (3-4 fields/region; 63x objective) of the apical surface of the anterior-ventral, anterior-dorsal, posterior-dorsal, and posterior-ventral region of lateral walls were acquired with Leica SP5 or SP8 white light laser scanning confocal microscope (Leica). The apical endings of B1 cells were manually counted using beta-Catenin to demarcate of the cell membrane and gamma-Tubulin to indicate basal bodies with Imaris Image Analysis software (v7.6-7.8, Bitplane). Data in Figs. 5D and S4A,B are expressed as density of B1 cells/mm² surface area at different time-points normalized to P21 and represented as mean +/− SD.
For identifications of apical B1 and non-apical B2 cells labeled by RCAS-GFP, V-SVZ whole-mounts from hGFAP::tva mice were immunostained for GFP, beta-Catenin and gamma-Tubulin or GFAP or VCAM-1 three, 14, or 28 days after injection with RCAS-GFP at P21 (n= 5, 3, and 7 mice, respectively). Images of GFP+ cells were acquired with a Leica SP5 or SP8 white light laser scanning confocal microscope and analyzed in 3D with Imaris Image Analysis software (v7.6-7.8, Bitplane).

Confocal images were taken with Leica SP8 white light laser scanning confocal microscope and BrdU intensities were determined using Leica LAS AF3 as gray scale values per pixel per region of interest (ROI). ROIs were defined as the area encompassing a BrdU⁺ nucleus with at least three BrdU patches. Normalized intensities were grouped into high, medium and low (above 80%, 50–80% and 50–20% relative to cortex, respectively. See Supplemental Information).

Upon BrdU administration at different ages, 3-4 images of 5-6 sections per OB were acquired with a Leica SP8 white light laser scanning confocal microscope. BrdU+ neurons were counted in 3D automated using Imaris Image Analysis software (v7.6-7.8, Bitplane). Data were expressed as densities (BrdU+ cells/mm² GCL) and are represented in Fig. 5D as mean +/− SD with n= 7, 4, 3, 3, 9, 4, and 3 mice for groups with BrdU administration at P21, P49, P77, P105, P116, P180, or P360, respectively. Compared to P21, the densities of newborn neurons born at P77-P360 declined, with statistical significance determined using one-tailed student’s t-test with equal variance (P21 vs P49, p= 0.24, n.s.; P21 vs. P77, p= 0.02 *; P21 vs. P105, p= 0.0015 **; P21 vs. P116, P180, or P360, p ≤ 0.0001 ***).