Samples of brain (0.1 ± 0.02 g) were homogenized in lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich). The supernatant was used for protein assay against a standard of bovine serum albumin (BSA) using a Bio-Rad Protein Assay [catalogue number (Cat. No.) 500–0001, Bio-Rad Laboratories, Inc., Munich, Germany)]. Next, 40 μg of proteins was separated using 8%–14% Sodium Dodecyl Sulfate (SDS) polyacrylamide gel and transferred onto nitrocellulose membranes. The nitrocellulose membranes were incubated with the following antibodies (Table 1 ): neuronal nuclei protein (Neu-N), neurofilament 200 KDa (NF), glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (IBA-1), aquaporin 4 (AQP4), intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). The blots were washed with PBS containing 0.5% Tween 20 (PBS-T) and then incubated with the horseradish-peroxidase-linked secondary antibodies (goat anti-rabbit IgG, Cat. No. A120–101P; or goat anti-mouse IgG, Cat. No. A90–116P, Bethyl Laboratory, Inc., Montgomery, TX, USA) at 1:5000 dilution for 60 min at room temperature, followed by visualization with an enhanced chemiluminescence kit (Lite Ablot® plus, Cat. No. EMP 011005, Euroclone, Life Sciences Division, Siziano, Italy). The images were acquired by the ChemiDoc imaging system (Bio-Rad Laboratories, Inc.). The β-actin protein (clone AC-74, Cat. No. A2228, Sigma-Aldrich Co., St. Louis, MO, USA) was used as a loading control at 1:3000 dilution in PBS-T overnight at 4°C. Band intensities were measured by densitometry with Nikon Imaging Software (NIS Elements) (Nikon, Florence, Italy). Blots are representative of three different experimental sessions.
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