Before extraction of Aβ from brain tissue, 10% (w/v) homogenates were prepared in tissue homogenization buffer (20 mm Tris base, pH 7.4, 250 mm sucrose, 1 mm EDTA, 1 mm EGTA) with 100 mm phenylmethylsulphonyl fluoride and protease inhibitors [protease inhibitors cocktail (Complete, Roche Diagnostic) plus pepstatin A] added immediately before homogenization, as we have previously published (Asuni et al., 2006 (link); Sadowski et al., 2006 (link); Scholtzova et al., 2008 (link)). For extraction of soluble Aβ, brain homogenates were thoroughly mixed with an equal volume of 0.4% diethylamine (DEA)/100 mm NaCl, then spun at 135,000 × g for 1 h at 4°C, and subsequently neutralized with 1/10 volume of 0.5 m Tris, pH 6.8. The samples were then aliquoted, flash-frozen on dry ice, and stored at -80°C until loaded onto ELISA plates. Similarly for extraction of the total Aβ, homogenates (200 μl) were added to 440 μl of cold formic acid (FA) and sonicated for 1 min on ice. Subsequently, 400 μl of this solution was spun at 100,000 × g for 1 h at 4°C. Then, 210 μl of the resulting supernatant was diluted into 4 ml of FA neutralization solution (1 m Tris base, 0.5 M Na2HPO4, 0.05% NaN3), aliquoted, flash-frozen on dry ice, and stored at -80°C until used for Aβ measurements.
The total and soluble Aβ levels were measured using a combination of mouse monoclonal antibody 6E10 (specific to an epitope present on amino acid residues 1-16 of Aβ) and two different rabbit polyclonal antibodies specific for Aβ40 (R162) and Aβ42 (R165), in a double-antibody sandwich ELISA as described previously (Sadowski et al., 2006 (link)). The optical density (OD) was measured at 450 nm. The relationship between OD and Aβ peptide concentration was determined by a four-parameter logistic log function. Nonlinear curve fitting was performed with the KinetiCalc program (Biotek Instruments) to convert OD of plasma to estimated concentrations. The assay was performed by an investigator blinded to group assignment. The levels of Aβ species are presented as μg of Aβ per g of wet brain, taking into account dilution factors introduced by multiple steps throughout the assay (brain homogenization and extraction procedures).
The total and soluble Aβ levels were measured using a combination of mouse monoclonal antibody 6E10 (specific to an epitope present on amino acid residues 1-16 of Aβ) and two different rabbit polyclonal antibodies specific for Aβ40 (R162) and Aβ42 (R165), in a double-antibody sandwich ELISA as described previously (Sadowski et al., 2006 (link)). The optical density (OD) was measured at 450 nm. The relationship between OD and Aβ peptide concentration was determined by a four-parameter logistic log function. Nonlinear curve fitting was performed with the KinetiCalc program (Biotek Instruments) to convert OD of plasma to estimated concentrations. The assay was performed by an investigator blinded to group assignment. The levels of Aβ species are presented as μg of Aβ per g of wet brain, taking into account dilution factors introduced by multiple steps throughout the assay (brain homogenization and extraction procedures).
