Anti aβ42

Right hemispheres from fresh frozen mouse brains were sectioned at 20 µm. Next, the sections were fixed with 4% paraformaldehyde and treated with pre-heated citrate buffer, pH 6.3, for 30 min followed by 70% formic acid for 5 min. Aβ was visualized with anti-Aβ40 (Agrisera, Umeå, Sweden), anti-Aβ42 1:1000 (700254, Thermo Fisher Scientific, USA), mAb158 or 3D6 1:1000 (in-house expression); activated astrocytes with anti-GFAP 1:200 (Abcam, Cambridge, UK); and microglia with antibodies against Iba1 (1:200) (Wako chemicals, Richmond, VA) and TREM2 1:200 (AF1729; R&D, Abingdon, UK). For colorimetric staining the Vector NovaRED™ horse radish peroxidase (HRP) substrate kit (Vector Laboratories, Burlingame, CA) was used for detection while for fluorescent staining Alexa secondary antibodies were used (Thermo Fisher Scientific, USA). For thioflavin S (ThS) staining, sections were pretreated in 95% and 70% ethanol (3 min in each), and quickly rinsed in water before they were incubated in 0.1% ThS for 10 min. Finally, the sections were briefly washed in 80% ethanol and water, dehydrated in ethanol, cleared in xylene and mounted with DPX.

Cell or mouse muscle and brain extracts were prepared in lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, halt proteinase inhibitor cocktail (ThermoFisher Scientific) and halt phosphatase inhibitor cocktail (ThermoFisher Scientific), and subjected to western blot analysis with anti-LC3 (Novus Biologicals, NB100-2220), anti-SQSTM1 (Abnova, H00008878-M01), anti-Aβ42 (Invitrogen; 700254), anti-APP (Biolegend; 803001), HRP-conjugated GFP antibody (Santa Cruz Biotechnology, sc9996), anti-HA (Cell Signaling Technology, C29F4), anti-ATG7 (Sigma Aldrich, A2856), anti-LDLR (Abcam, ab52818), anti-LRP1 (Abcam, ab92544), and anti-ACTB/β-actin-HRP (Santa Cruz Biotechnology, sc47778 HRP) antibodies. The band intensity was analyzed using the ImageJ software.