Brains were cut into 40 µm coronal sections on a microtome (LEICA SM2010; Leica Biosystems, Inc., Deer Park, IL, USA) and stored at 4 °C in a cryoprotective solution, then processed for immunohistochemistry until required. Immunohistochemistry for Ki67 detection (1:500; Abcam, Waltham, MA, USA), BrdU (1:500; BD-Pharmigen®, Franklin Lakes, NJ, USA), DCX (1:250; Abcam, MA, USA), c-fos (c-fos 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), or Iba-1 (1:1000; Wako Chemicals, Co., Richmond, VA, USA) was performed using the peroxidase method in series of every sixth section, as previously reported [41 (link),45 (link),46 (link),52 (link)]. All Ki67-, BrdU-, DCX-, c-fos-, and Iba-labeled cells were counted throughout the rostro-caudal extension of the hippocampus, using a light microscope (Leica microsystems, Wetzlar, Germany, Germany). In the DG, the quantification of BrdU-, Ki67-, DCX-, c-fos, and Iba-labeled cells was limited to the granular cell layer (GCL) and sub-granular zone (SGZ). The latter region was defined as a band, limited by three nuclei down from the apparent border between the GCL and the hilar region (H). To obtain the estimated total number of Ki-67-, BrdU-, DCX-, c-fos, or Iba-labeled cells, the resulting number of positive cells for each marker was multiplied by six [41 (link),52 (link),53 (link),54 (link),55 (link)].
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