Horizontal sections (8 μm thick) of frozen brain were taken between 1.5 and 2.5 mm deep (dorsal) and fixed. For bromodeoxyuridine (BrdU) staining, the sections were fixed in acetic acid, 5%, and ethanol, 95%, for 10 minutes at −20°C, followed by hydrogen peroxide, 0.3%, in methanol for 15 minutes at room temperature (mouse anti-BrdU, 1:20; Dako, Glostrup, Denmark; and neuronal nuclei antibody, 1:50; Chemicon International, Inc, Temecula, California). For ionized calcium binding adaptor molecule (IBA-1), glial fibrillary acidic protein (GFAP), and major histocompatibility complex (MHC) class II, the sections were fixed in formaldehyde, 4% (IBA-1, 1:200; Wako Chemicals, Osaka, Japan; GFAP, 1:200; Dako; and MHC class II clone IBL 5/22, 1:50; Chemicon International, Inc). For intercellular adhesion molecule 1 (ICAM-1), sections were fixed in fresh cold acetone (1:200; Dako) and in paraformaldehyde, 4% (calbindin, 1:200; Biotest Ltd, Kfar Saba, Israel). Following incubation with the primary antibody (according to the manufacturers' recommendations), sections were incubated with a goat-antimouse IgG secondary antibody conjugated to Alexa Fluor 488 (dilution, 1:100; Molecular Probes, Inc, Eugene, Oregon) for 50 minutes at room temperature or with antirabbit cy5 (dilution, 1:100; Molecular Probes, Inc). Counterstaining was performed with 4",6-diamidino-2-phenylindole (DAPI). For the expression of MHC class II and calbindin, to measure the thickness of the CA1 neuronal layer, and to determine BrdU-immunoreactive neuronal cells, images of a total of 16 microscopic fields (4 fields for each mouse brain, at magnification ×100) containing cerebellum or hippocampus were obtained under identical conditions from each group and stored using a video camera. The positively stained cells (BrdU, MHC II) or Purkinje cells (calbindin) and the positively stained blood vessels (ICAM-1) were counted and the thickness of the CA1 layer was measured. For hematoxylin-eosin staining, sections were incubated in hematoxylin for 20 minutes at room temperature and then in eosin, 2%, for 2 minutes at room temperature (Sigma).