Aβ1 16 6e10

The human and mouse brain extracts were treated with increasing concentrations (0, 25, 50, and 100 µg/mL) of proteinase K (PK) (Nacalai Tesque, Inc., Kyoto, Japan) for 1 h at 37 °C. PK treatment was blocked by adding NuPage LDS Sample Buffer (Thermo Fisher Scientific, Inc., Massachusetts, USA) and NuPage Sample Reducing Agent (Thermo Fisher Scientific, Inc.), and then incubated for 10 min at 70 °C. The samples were analyzed by Western blotting using NuPage 4–12% Bis–Tris Gel using NuPage MES running buffer (Thermo Fisher Scientific, Inc.) and antibodies against Aβ_1–16 (6E10, 1:2,000; BioLegend) as the primary antibodies.

Fluorescent immunolabeling was performed using a standard indirect technique as described previously [22 (link)]. Brain sections were stained with primary antibodies against: ionized calcium binding adaptor molecule 1 (IBA1; 1:1000; 019-19741, Wako and ab5076, Abcam), CD68 (1:200; BioRad) glial fibrillary protein (GFAP; 1:1000; Abcam), NeuN (1:1000; Millipore), OLIG2 (1:200; Abcam), Aβ1–16 (6E10; 1:1000; Biolegend), and anti-lysosomal associated membrane protein 1 (LAMP1; 1:200; Santa Cruz Biotechnologies). For Amylo-Glo staining (TR-300-AG; Biosensis), tissue sections were washed in 70% ethanol 1 × 5 min, followed by a 1 × 2 min wash in distilled water. Sections were then placed in a 1% Amylo-Glo solution for 1 × 10 min then washed with 0.9% saline for 1 × 5 min and distilled water for 1 × 15 s before continuing fluorescent immunolabelling. For Thioflavin-S (Thio-S) staining, tissue sections were placed for 1 × 10 min incubation in 0.5% Thio-S (1892; Sigma-Aldrich) diluted in 50% ethanol. Sections were then washed 2 × 5 min each in 50% ethanol and one 10-min wash in 1xPBS before continuing with fluorescent immunolabelling.
High resolution fluorescent images were obtained using a Leica TCS SPE-II confocal microscope and LAS-X software. For confocal imaging, one field of view (FOV) per brain region was captured per mouse unless otherwise indicated.