A 31577

Mice were anesthetized and intracardially perfused with ice-cold PBS and 4% paraformaldehyde. Brains were harvested and post-fixed for 2–4 h at 4°C. Brains were washed in PBS and cryoprotected in 30% sucrose in PBS. Coronal sections (15 μm thick) were collected with a freezing microtome (Leica). The sections were washed in PBS and then blocked in blocking buffer (5% BSA and 0.2% Triton X-100) for 1 h at room temperature, and then subsequently incubated with primary antibodies against human Aβ (6E10, BioLegend, 803014, 1:400) and LAMP1 (Abcam, ab24170, 1:200) overnight at 4°C, and appropriate fluorescence-conjugated secondary antibodies (Thermo Fisher Scientific, A-11008, A-11005 or A-31577, 1:500) for 1 h at room temperature in the dark. Confocal images were acquired using the A1R (Nikon) or FV1000MPE-B (Olympus) confocal microscope. All images were processed with ImageJ to calculate the area of Aβ plaques and dystrophic neurites.

Immunohistochemistry was performed on paraffin-embedded mouse brain sagittal sections (5 µm) after deparaffinization and antigen retrieval. Triple immunostaining for NeuN + , GFAP + , IBA1+ cells was performed using a mouse anti-NeuN antibody for neurons (1:400; MAB377, Millipore), a rabbit anti-IBA1 antibody for total microglia (1:300; 016-20,001, WAKO) and a mouse anti-GFAP antibody conjugated with Alexa Fluor 488 (1:500; MAB3402X, Millipore) for astrocytes^{76 (link)}. Alexa Fluor 635 anti-rabbit (1:600; Alexa Fluor 555 anti mouse antibody (1:600; A31577 and A31570, Thermo Fisher Scientific) were used as secondary antibodies.