Fractionation and Quantification of Amyloid-β

Soluble and insoluble ingredients of Aβ were detected as previously reported (10 (link), 72 (link)). The frozen tissues were homogenized in 15 volumes (w/v) of TBS homogenization buffer, which contained phosphatase and protease inhibitor cocktails (Thermo Fisher Scientific), and centrifuged at 100,000g for 1 hour at 4°C. The supernatant fraction was collected as TBS-soluble fraction, and the sediment fraction was resuspended in 15 volumes (w/v) of 1% Triton X-100/TBS (TBS-X). And then the samples were incubated on ice for 30 minutes followed by centrifuging at 100,000g for 1 hour at 4°C. The supernatant fraction was collected as TBS-X–soluble fraction, and the sediment fraction was resuspended with 15 volumes (w/v) of 70% FA. After the samples were centrifuged at 100,000g for 1 hour at 4°C, the supernatant fraction was neutralized by 20 volumes 1 M Tris base (pH 11), which was named FA-soluble fraction. The protein concentration of TBS-soluble fraction and TBS-X–soluble fraction was measured by BCA protein assay kit (Thermo Fisher Scientific). And protein concentration of FA-soluble fraction was measured by a Bradford protein assay kit (Beyotime). The Aβ₄₀ and Aβ₄₂ levels were quantified with Quantikine ELISA Human Amyloid β aa1-40/aa1-42 immunoassay kits (R&D Systems) according to the manufacturer’s protocol.