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).