Anti brdu

First, free-floating sections were washed in PBS three times, followed by incubation in blocking buffer (0.1% Triton X-100, 1% bovine serum albumin, 5% normal goat serum in PBS) for 1 h. Then sections were incubated with primary antibody overnight at 4°C, washed for three times in PBS, and then incubated with secondary antibody for 2 h at 4°C. After 4′,6-diamidino-2-phenylindole (DAPI) treatment for 2 min, sections were washed again with PBS three times and mounted with Fluoromount-G (SouthernBiotech, Birmingham, AL). For BrdU incorporation, sections were treated before standard blocking protocol as follows: incubation with 2N HCl was performed at room temperature for 1 h, followed by soak in 0.1 m (pH 8.4) borate buffer for 10 min. After three washes in PBS, sections were processed according to standard protocol in blocking solution. The primary antibodies and their final concentrations used in the experiments were as follows: anti-BrdU (1:200, rat; AbD Serotec, Raleigh, NC), anti-pS6 (1:200, rabbit; Cell Signaling Technology, Danvers, MA), anti-GFP (1:1000, chicken; Abcam, Cambridge, MA), and anti-Sox2 (1:1000, goat; R&D Systems, Minneapolis, MN). Secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA) and applied in a 1:1000 dilution.

Immunohistochemistry was conducted as previously described [31 (link)]. Briefly, 14- months old mice were overdosed with pentobarbital and transcardially perfused with 0.9% saline solution. Brains were removed and hemispheres were separated. Hemi-brains were placed in 4% paraformaldehyde overnight prior to cryoprotection in increasing sucrose gradients (10%, 20% and 30% each day). Hemi-brains were sectioned horizontally on a freezing microtome at a thickness of 25 µm and stored in phosphate-buffered saline containing 0.02% NaN₃ at 4 °C until staining. Six sections containing the hippocampus were selected for staining. Immunofluorescent staining was completed using anti-BrdU (1:200; AbD Serotec) and anti-NeuN (1:100; Millipore) primary antibodies with Alexa Fluor 594 and 633 secondaries (1:1000; Invitrogen). Sections were then mounted in glass slides and allowed to dry overnight, followed by cover-slipping the next day. Slides were scanned into digital images using a Zeiss AxioScan.Z1 slide scanner. Positive staining was determined visually based on morphology and co-localization of BrdU with NeuN and counted by hand for both the dentate gyrus and subventricular zones.