Human aβ40 or aβ42 standards

Aβ levels were quantified using a sensitive sandwich ELISA as previously described (Blurton-Jones et al., 2009 (link)). In brief, immulon 2HB flat-bottom wells (Thermo Scientific) were coated overnight at 4°C with monoclonal anti-Aβ_1–16 (generous gift of Dr. W. van Nostrand, Stony Brook University) at 30 µg/mL in 0.1M carbonate buffer (pH 9.6). Wells were then blocked with 3% BSA in PBS at 37° C for 3 h. Next, plates were washed and wells loaded in triplicate with brain homogenates or human Aβ₄₀ or Aβ₄₂ standards (EMD Millipore) diluted in capture buffer (20 mM sodium phosphate, 0.4M NaCl, 2 mM EDTA, 0.3% BSA, 0.05% CHAPS, and 0.05% Na azide, pH 7.0) and incubated overnight at 4°C. Plates were washed again and then probed overnight at 4°C with either biotinylated monoclonal anti-Aβ₄₀ or anti-Aβ₄₂. After washes, wells were incubated with streptavidin-HRP (Thermo Scientific) for 3 h at 37° C. Plates were then developed with Ultra TMB-ELISA substrate (Thermo Scientific) followed by 0.8M O-phosphoric acid to stop the reaction. Plates were then read at 450 nm using a microplate photometer (Labsystems).