Immunohistochemistry for amyloid β

The brains of mice given the intravenous administration were fixed in 4% paraformaldehyde phosphate buffer solution (Wako Pure Chemical). The fixed tissues were dehydrated with ethanol (Wako Pure Chemical) for 24 h, transferred into xylene (10 min three times), and embedded in paraffin (30 min three times at 60°C). The paraffin-embedded tissue blocks were cut into 4-μm thick sections with a sliding microtome (Nippon Optical Works Co., Ltd., Tokyo, Japan) and extended on Matsunami-Adhesive Silane-coated micro slide glasses (Matsunami Glass Industry, Ltd., Osaka, Japan) at 60°C for 24 h. The sections were deparaffinized with xylene and ethanol (100%, 90%, and 70%). After washing twice in water, 70% formic acid solution was dispensed on to the sections, and they were incubated at room temperature for 5 min. The sections were washed with water (5 min, three times) and 0.2% Tween 20 in PBS (10 min), and then stained with anti-Aβ antibody 6E10 (1:100 dilution; Covance Inc., Princeton, NJ, USA) using a M. O. M.TM Immuno detection Peroxidase Kit (Vector Laboratories Inc., Burlingame, CA, USA), according to the manufacture’s protocol. As a chromogen, 3,3’-diaminobenzidine (Wako Pure Chemical) at a concentration of 0.5 mg/ml in PBS with 0.005% hydrogen peroxide was used. After staining, the sections were washed with water, dehydrated with ethanol (70%, 90%, and 100%) and xylene, and then mounted with mounting medium (Daido Sangyo Co., Ltd., Tokyo, Japan). The sections were observed with a microscope BZ-8100 (Keyence Corp., Osaka, Japan). Aβ plaques in each section were detected using ImageJ software (National Institutes of Health, Bethesda, MD, USA).The number of Aβ plaques in the total section area, the ratio of Aβ plaque area to the whole section area and the average size of Aβ plaque of each section were calculated.