Biotinylated goat anti rabbit igg and streptavidin peroxidase complex

Immunohistochemistry was performed under the same conditions for all groups. The brain sections were treated with 0.3% hydrogen peroxide in PBS for 30 min and then with 10% normal goat serum (Vector Laboratories, USA) in 0.05 M PBS for 30 min at room temperature. The sections were then incubated with rabbit anti-GR antibody (1 : 1,000 dilution, clone M-20; Santa Cruz Biotechnology, USA) overnight at room temperature, and subsequently exposed to biotinylated goat anti-rabbit IgG and streptavidin peroxidase complex (1 : 200 dilution; Vector Laboratories) at room temperature. Antibody binding was visualized by staining with 3,3'-diaminobenzidine in 0.1 M Tris-HCl buffer (pH 7.2). The sections were mounted on gelatin-coated slides and dehydrated. Negative control samples were incubated with pre-immune serum instead of primary antibody to measure immunostaining specificity.

According to our published procedure (33 (link)), in brief, mice (n=7 at each point in time) were anesthetized with pentobarbital sodium (40 mg/kg, JW Pharmaceutical Co., Ltd., Seoul, Korea) and perfused with 4% paraformaldehyde. Brain tissues were serially sectioned in a cryostat (Leica, Wetzlar, Germany) into 30-µm coronal sections. The sections were incubated with diluted rabbit anti-NF-H (1:1,000; Chemicon International, Inc., Temecula, CA, USA), NF-M (1:1,000; Chemicon International, Inc.) or NF-L (1:1,000; Chemicon International, Inc.), and exposed to biotinylated goat anti-rabbit IgG and streptavidin peroxidase complex (Vector Laboratories, Inc., Burlingame, CA, USA). Finally, they were visualized with 3,3′-diaminobenzidine tetrachloride (Sigma-Aldrich; Merck KGaA).

To ensure that the immunohistochemical data were comparable between groups, the sections were carefully processed under the same conditions. The sections were hydrated and treated with 0.3% hydrogen peroxide (H₂O₂) in PBS for 30 min. For antigen retrieval, the sections were placed in 400 mL jars filled with citrate buffer (pH 6.0) and heated in a microwave oven (Optiquick Compact, Moulinex, UK) operating at a frequency of 2.45 GHz and 800 W power setting. After three heating cycles of 5 min each, slides were allowed to cool at room temperature and were washed in PBS. After washing, the sections were incubated with 5% non-fat dry milk in PBS containing 0.1% Tween 20 for 45 min to reduce the background staining. Sections were incubated successively in 10% normal goat serum in PBS for 30 min, and in diluted rabbit anti-GLUT3 (diluted 1:50, SantaCruz Biotechnology, Santa Cruz, CA, USA) for 48 h at 4℃. Thereafter, they were exposed to biotinylated goat anti-rabbit IgG and streptavidin peroxidase complex (diluted 1:200, Vector, Burlingame, CA, USA) and visualized with 3,3'-diaminobenzidine tetrahydrochloride (Sigma) in 0.1 M Tris-HCl buffer (pH 7.4).