Immunohistochemical Analysis of Ischemic Hippocampus

In order to study neuronal damage and glial activation in the ischemic hippocampi, immunohistochemistry was performed according to our published protocol [27 (link),28 (link)]. We used antibodies as follows: (1) neuronal nuclei (NeuN) for neurons; (2) glial fibrillary acidic protein (GFAP) for astrocytes; (3) ionized calcium-binding adapter molecule 1 (Iba-1) for microglia. In brief, the brain sections were treated with 0.3% hydrogen peroxide (H₂O₂) in PBS (pH 7.4) for 40 min, followed by 10% normal rabbit serum (Vector Laboratories, Inc., Burlingame, CA, USA) in PBS for 40 min. Furthermore, these pretreated sections were reacted with mouse anti-NeuN (diluted 1:1000) (Chemicon, Temecula, CA, USA) for neurons, mouse anti-GFAP (diluted 1:800) (Chemicon, Temecula, CA, USA) for astrocytes and rabbit anti-Iba-1 (diluted 1:800) (Chemicon, USA) for microglia. Subsequently, these sections were exposed to biotinylated goat anti-mouse immunoglobulin G (IgG) or goat anti-rabbit IgG and streptavidin peroxidase complex (diluted 1:200, respectively) (Vector, Burlingame, CA, USA), and they were visualized with 3,3’-diaminobenzidine (in 0.1 M Tris HCl buffer (pH 7.4). Finally, the immunoreacted sections were prepared for examination of each immunoreactivity under a light microscope.