Tissue was fixed via transcardial perfusion with 4% paraformaldehyde, under ketamine/xylazine anesthesia. Brains were then isolated and post-fixed (4% paraformaldehyde for 4 h at 4°C) followed by cryoprotection with 30% sucrose. Coronal sections (20 or 40 μm) through the dorsal (stereotaxic coordinate AP: −1.40 ~ −2.20) hippocampus were prepared using a freezing microtome.
For immunohistochemistry, sections were washed with phosphate-buffered saline (PBS) and incubated in 0.3% hydrogen peroxide/PBS for 20 min to eliminate endogenous peroxidase activity. After several washes with PBS, sections were blocked with 10% normal goat serum or 3% normal horse serum in PBS, then incubated overnight at 4°C with the following antibodies: rabbit anti-pCREB (1 : 1000, Cell Signaling, Danvers, MA, USA), rabbit anti-Ki-67 (1 : 2000, Vector labs, Burlingame, CA, USA), goat anti-doublecortin (1 : 1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-Sox-2 (1 : 1000, Santa Cruz Biotechnology), goat anti-MSK1 (1 : 1000, Santa Cruz Biotechnology: the specificity of this antibody was verified in Karelina et al. (2012). The ABC labeling method (Vector Labs) followed by nickel-intensified DAB development (Vector Labs) was used to visualize the signal. Images were acquired using a 16-bit digital camera (Micromax YHS 1300; Princeton Instruments, Trenton, NJ, USA) mounted on a Leica DM IRB microscope (Nussloch, Germany). For BrdU staining, sections were incubated in 2XSSC/50% formamide for 2 h at 65°C, followed by incubation in 2 N HCl at 37°C for 1 h. After washing with 0.05 M borate buffer (pH 8.5) for 10 min and washing with PBS, sections were blocked with 10% normal goat serum in PBS and incubated at 4°C with a rat anti-BrdU antibody (1 : 400, Accurate Chemical, Westbury, NY, USA). After washing with PBS, sections were incubated with horseradish peroxidase-conjugated anti-rat IgG (1 : 400, Jackson Immunoresearch, West Grove, PA, USA) for 2 h at 22°C and developed using nickel-intensified DAB.
For immunofluorescence labeling, sections were washed with PBS, DNA was denatured as noted above and blocked with 10% normal goat serum or 5% normal horse serum in PBS, followed by overnight incubation at 4°C with rat anti-BrdU antibody (1 : 400), mouse anti-NeuN antibody (1 : 1000, Millipore, Billerica, MA, USA), goat anti-doublecortin antibody (1 : 500), rabbit anti-MSK1 antibody (1 : 1000), or goat anti-SOX2 antibody (1 : 500). After several washes, sections were incubated (2 h at 22°C) with secondary antibodies conjugated with Alexa 488 or Alexa 594 (1 : 1000, Invitrogen, Carlsbad, CA, USA), then mounted with Cytoseal (Richard-Allan Scientific, Kalamazoo, MI, USA). Fluorescence images were captured using a Zeiss 510 Meta confocal microscope (Carl Zeiss Ag, GmbH, Oberkochen, Germany) (2-μm-thick optical section). Cresyl violet staining was performed as described in Choi et al. (2007).