Mn1020b

The ELISA plates were coated with 20-μg/ml coating antibody overnight at 4°C. The next day, the plates were washed in PBS for five times, and mouse brain lysates were diluted in sample buffer (human tau and ptau sample buffer: 0.25% BSA in PBS with 300 μM Tris, pH 7.4; human apoE sample buffer: 0.5% BSA in 0.025% PBS-Tween 20 [PBST]; both buffers were supplemented with protease inhibitors) and loaded into the plates in duplicates. The plates were incubated at 4°C overnight. On the third day, the plates were washed in PBS, followed by addition of the detection antibody and incubation at 37°C for 90 min. The plates were then washed with PBS and incubated with streptavidin-poly-horseradish peroxidase-40 for 90 min at RT. Plates were then washed and developed with Super Slow ELISA 3,3′,5,5′-Tetramethylbenzidine (Sigma) and read at 650 nm. For FA fractions, samples were neutralized with 3 M Tris buffer (pH 8.0) before loading into the plate. Tau5 (gift from L. Binder, Northwestern University, Chicago, IL), HJ14.5 (in house anti-ptau antibody) and HJ15.6 (in house anti-human apoE antibody) were used as coating antibodies, and biotinylated HT7 (MN1000B; Thermo Fisher Scientific), biotinylated AT8 (MN1020B; Thermo Fisher Scientific), and biotinylated HJ15.4 (in house anti-human apoE antibody) were used as detection antibodies for human tau, ptau, and human apoE ELISAs, respectively.