Brain tissues were fixed in 4% neutral buffered paraformaldehyde (pH = 7.4) and embedded in paraffin. The paraffin sections (5 μm) were deparaffinized in xylene and rehydrated in ethanol; then, antigen retrieval was performed by incubating in oiled 0.01 M sodium citrate buffer (pH = 6) and blocking endogenous peroxidase activity using 10% H2O2. To reduce nonspecific staining, the slides were immersed in Protein Block, Serum Free (Dako, Tokyo, Japan) for 15 min. For immunohistochemical staining, the sections (5 μm) were incubated with anti-BDNF (1:100, Abcam, Cambridge, UK), anti-GR (1:400, Cell Signaling Technology, Danvers, MA, USA) or anti-doublecortin (1:1000, Abcam, Cambridge, UK) and then incubated with biotinylated goat anti-rabbit IgG and streptavidin peroxidase complex (Nichirei Biosciences Inc., Tokyo, Japan) for 30 min at room temperature, stained with diaminobenzidine and then counterstained with hematoxylin. A light microscope (Olympus, BX50, Tokyo, Japan) connected to a digital camera was used for examining and photographing the slides. For quantification of doublecortin-positive cells in the DG of both hippocampal lobes, the number of immunopositive cells was counted in 10 randomly selected fields of sections, original magnification ×400, as previously described [72 (link)].
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