2.10 Immunostaining
The mouse brains were cut on a cryostat (Leica, CM1850) at a thickness of 10 μm. A series of three equally spaced brain sections (~1 mm apart) were used for each type of stain. The slides or cells were fixed with 4% paraformaldehyde for 10 min, followed by permeabilized with 0.2% Triton X-100 for 10 min at room temperature. After blockage with 5% BSA (Sigma-Aldrich) for 1 h, sections or cells were incubated with mouse anti-Aβ (Santa Cruz, sc-28365, 1: 400), rabbit anti-p-APP (Cell Signaling Technology, 6986, 1: 100), rabbit anti-human Lf (Abmart, T59526, 1: 100), mouse anti-GFAP (Cell Signaling Technology, 3670, 1: 200) and mouse anti-NeuN (Cell Signaling Technology, 94403, 1: 200) overnight at 4 °C. The secondary antibodies, goat anti-rabbit-Ig G Alexa 555 (Themo Fisher Scientific, A32732, 1:400), goat anti-mouse-Ig G Alexa 555 (Themo Fisher Scientific, A21422, 1:400), goat anti-rabbit-Ig G Alexa 488 (Themo Fisher Scientific, A11008, 1:400) and goat anti-mouse-Ig G Alexa 488 (Themo Fisher Scientific, A32723, 1:400) were employed. Images of Aβ staining in the half mouse brains were captured by fluorescent microscope (Nikon, NI-SH-E), and the other images were obtained using a confocal laser microscope (Nikon, A1). The quantifications of the numbers and areas of Aβ-positive plaques, as well as the fluorescent intensities of Aβ and p-APP were both performed using Image J software. At least twenty-five cells were measured to evaluate the Aβ index and p-APP index in a single N2a-sw cell (Fan et al., 2019).