Thioflavin S staining

AβPP/PS1 mice were killed after the behavior test. The mice were deeply anesthetized with chloral hydrate, and then were immediately cardiac perfused with 0.9% saline solution followed by 4% paraformaldehyde in 0.1 M PBS (pH: 7.4). After the perfusion, the brains of the mice were excised and bisected, and a hemibrain was postfixed overnight at 4°C. The brain tissue was then incubated in 30% sucrose at 4°C until equilibration (6 mice/group). Thirty micrometer coronal sections were cut by a freezing microtome (CM1850; Leica, Germany) and stored at −20°C. For thioflavin S staining, six serial sections with an interval of 50 μm were taken from the cortex and the hippocampus per mouse. Brain sections were incubated in 0.5% Thioflavin S (Sigma-Aldrich; St. Louis, Missouri, USA) dissolved in 50% ethanol for 5 min, and then washed twice with 50% ethanol for 5 min each time. The brain sections were washed once with tap water for 5 min, and then mounted with mounting medium. The green fluorescence-stained plaques were observed under a fluorescent microscope. The staining was analyzed by the image analyzing system, Image pro plus 6 (Media Cybernetics; Rockville, Maryland, USA).