Quantification of Aβ by immunohistochemistry

Quantification of cerebral amyloid-β (Aβ) load was performed as described in detail in ref. [48]. Briefly, 5xFAD and control mice were transcardially perfused with ice-cold saline and followed by 4% PFA solution. The brains were post-fixated in 4% PFA for 4 h at 4 °C and subsequently transferred to first a 30% sucrose solution and then an antifreeze solution and stored at −20 °C. Frozen tissue was cut on a microtome (35 µm) (Leica SM 2000R). Sections were preheated, rinsed, and incubated with the primary antibody (mouse anti-Aβ, 1:1000) overnight. Sections were subsequently incubated with the secondary antibody (goat anti-mouse, 1:400) for 2 h at 20 °C, before transfer to a solution containing mouse StreptAvidin (1:1000) for 2 h. Finally, sections were incubated for approximately 3 min in Ni-enhanced DAB solution. Sections were automatically scanned (Hamamatsu NanoZoomer XR) and analyzed using Adobe Photoshop. Aβ plaques were measured using the color range command. The final values were obtained by dividing the Aβ plaques area by the total area of the section.