BrdU Administration and Immunohistochemistry

BrdU (Sigma-Aldrich) was prepared as a 20-mg/mL solution in sterile saline and injected intraperitoneally into 10–11-week- old mice at a dose of 100 mg/kg every 12 h for three doses, and mice were sacrificed 2 days after the last injection. Brains were perfused with ice-cold PBS, fixed in 4 % paraformaldehyde overnight, and then incubated in 30 % sucrose. For immunohistochemistry of BrdU and doublecortin (DCX), 40-μm thick sections were subjected to antigen retrieval procedure as previously described [12]. Briefly, sections were incubated in deionized formamide/1XSSC solution for 2 h at 65 °C, followed by incubation in 2 N HCl for 30 min at 37 °C. Sections were neutralized with 0.1 borate buffer (pH 8.5) and washed six times, 10 min each wash, in TBS. Brain sections were blocked using 5 % normal donkey serum in 1XTBS with 0.25 % Triton X-100 (1XTBST) for 2 h at room temperature, followed by incubation of primary antibodies for 72 h in blocking solution at 4 °C. Primary antibodies used were rat anti-BrdU (1:400, Accurate Chemical) and goat anti-DCX (1:400, Santa Cruz). Secondary antibodies used were donkey anti-rat Alexa Fluor 488, donkey anti-goat Alexa Fluor 594, and donkey anti-rabbit Alexa Fluor 647 (1:250, Invitrogen). Brain sections were counterstained with DAPI (1:25,000, Invitrogen) for 5 min before mounting with polyvinyl alcohol mounting media with DABCO® (PVA-DABCO) and analyzed using confocal UV LSM 500 Zeiss microscope. Quantification of positive BrdU+ and DCX+ cells in the subgranular layer and subventricular zone was performed by stereology (StereoInvestigator 8; MBF Bioscience) or by ImageJ with cell counter plug-in, respectively, as previously described [12, 13]. At least three mice per genotype were used for the analysis with statistics performed using the Student’s t test.