Anti rat cy3 conjugated secondary antibody

Immunofluorescence for the BrdU-positive and NeuN-positive cells in the hippocampal dentate gyrus was performed, according to the previously described method (Shin et al., 2013 (link)). The sections were first permeabilized by incubation in 0.5% Triton X-100 in PBS for 20 min, then pretreated in 50% formamide-2 X standard saline citrate (SSC) at 65°C for 2 h, denatured in 2 N HCl at 37°C for 30 min, and rinsed twice in 100 mM sodium borate (pH 8.5). After washing in PBS for 15 min at 27°C, the sections were followed by wash for 10 min three times at room temperature. The sections were incubated overnight with rat anti-BrdU antibody (1:200; Abcam, Cambridge, Cambridgeshire, UK) and mouse anti-NeuN antibody (1:400; Chemicon, Temecula, CA, USA). The sections were next incubated for 2 h with cy3-conjugated anti-rat secondary antibody (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for the BrdU antibody and fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (1:200; Jackson Immuno Research Laboratories) for the NeuN antibody. The sections were then mounted on gelatin-coated glass slides, and the cover slips were mounted using fluorescent mounting medium (Dako Cytomation, Carpinteria, CA, USA). The slides of the fluorescent images were captured using a confocal laser scanning microscopy (LSM-700; Carl Zeiss, München-Hallbergmoos, Germany).