After the marmosets had performed all the experiments, including the pre- and post-lesion sessions, they were deeply anesthetized and transcardially perfused with 4% paraformaldehyde in phosphate buffer (pH 7.4). The fixed brains were removed from the skull, postfixed in the same fresh fixative overnight at 4°C, and placed into 0.1 M phosphate buffer (pH 7.4) containing 30% sucrose. The brains were then cut along the coronal plane into 50 μm thickness slices using a freezing microtome. One section out of six was immediately mounted for thionin staining. For immunohistochemistry, adjacent sections were incubated with a mouse monoclonal antibody for glial fibrillary acidic protein (1:1,500 dilution; Sigma-Aldrich, St. Louis, MO, USA) or a rabbit polyclonal antibody for Iba-1 (1:4,000 dilution; WAKO Pure Chemical Industries, Osaka, Japan). Secondary biotinylated anti-mouse (1:200 dilution; Vector Laboratories, Burlingame, CA, USA) or biotinylated anti-rabbit (1:200 dilution; Vector Laboratories) antibodies were also used. Immunoreactive signals were visualized using the ABC Staining Kit (Vector Laboratories) with 3,3'-diaminobenzidine. All stained images were acquired using an inverted microscope (BZ-X700, Keyence, Osaka, Japan).
Free full text: Click here
