Quantification of Amyloid-beta Peptides

Levels of Aβ₄₀ and Aβ₄₂, corresponding to total Aβ load, was measured in the formic acid (FA) treated brain homogenates, as previously described [25 (link)]. In brief, 96-well plates were coated overnight with 100 ng/well of rabbit polyclonal anti-Aβ₄₀ or anti-Aβ₄₂ (custom made by Agrisera) and blocked with 1% BSA in PBS. FA extracts from frontal cortex were neutralized with 2 M tris and diluted 2000× for Aβ₄₀ and 200× for Aβ₄₂ analysis, then incubated overnight, followed by detection with biotinylated mAb1C3 (0.5μg/ml) [26 (link)] and streptavidin-HRP (Mabtech AB). Signals were developed with K Blue Aqueous TMB substrate (Neogen Corp.) and read with a spectrophotometer at 450 nm. All dilutions were made in ELISA incubation buffer (PBS with 0.1% BSA, 0.05% Tween20 and 0.15% Kathon).