Immunohistochemical Analysis of Neurogenesis

The brains of mice were removed and fixed in phosphate-buffered 4% paraformaldehyde (pH 7.4). Then, the fixed samples were sectioned in the coronal plane at 40 μm thickness using a cryomicrotome (Leica, Wetzlar, Germany). The obtained sections were incubated with 0.01 M sodium citrate buffer (pH 6.0) for 30 min at 90°C and then washed with Tris-buffered saline with Tween (TBST). Next, the sections were blocked in 10% goat serum for 1 h and then incubated with anti-BrdU (1:1,000), anti-DCX (1: 500), anti-Sox-2 (1:200), or anti-p-CREB (1:1,000) overnight at 4°C. Staining was revealed using biotinylated secondary antibodies and avidin-biotin complex (ABC) peroxidase standard staining kit (Thermo Fisher Scientific) with diaminobenzidine (DAB, Thermo Fisher Scientific). Immunopositive cells were quantified using Fiji ImageJ software (https://fiji.sc/). The total number of BrdU, DCX, and Sox-2-immunopositive cells (BrdU⁺, DCX⁺, and Sox-2⁺) per DG was estimated based on the method described previously (17 (link)), while p-CREB immunoreactivity in the granule cell layer (GCL) of the DG was quantified by using the IHC Profiler plugin for ImageJ according to the procedures reported by Varghese et al. (18 (link)). For each marker, 5 sections of the DG from an individual animal were randomly selected and analyzed by the same observer, who was unware of animal assignments.