These mechanistic insights lay the foundation for the generation and engineering of safe and highly efficacious Aβ antibodies for DNA Synthesis inhibitor the removal of existing plaque in Alzheimer’s patients. This is an important goal since biochemical and neuroimaging data demonstrate the presence of extensive plaque deposition in AD patients some 10 years prior to first memory complaint (Jack et al., 2010; Morris and Price, 2001; Price et al., 2009)

and indeed by the time of diagnosis, plaque deposition is already reported to be at or near maximal levels. Multiple colonies of PDAPP mice were utilized for the current studies. PDAPP line 1683 heterozygous for the APPV717F transgene was maintained on a mixed outbred background as previously described (Johnson-Wood et al., 1997). The phenotype of the PDAPP line 1683 colony began to change wherein plaque deposition initiated at later ages, and there was a dramatic increase in the variability of deposited Aβ in middle-aged mice (8–14 months old). A new PDAPP colony (line 6042) was established through an inbreeding exercise wherein mice were

inbred from selected litters that maintained decreased variability in both soluble and insoluble Aβ. The plaque deposition phenotype of the inbred PDAPP line 6042 was similar to the originally described PDAPP colony (Games et al., 1995). All experiments were performed in accordance with the Institutional Animal Care and Methisazone Use Guidelines for Eli Lilly. Frozen brain tissue of an AD Y-27632 order patient was embedded in M-1 Embedding Matrix at −20°C, sectioned to 20 μm, mounted on poly-D-lysine-coated cover glass (15 mm), and placed in 24-well tissue culture plates. Sixty four consecutive sections were positioned in the same order

as sectioned and were incubated with or without antibodies (10 μg/ml, 500 μl, 1 hr, room temperature). The control IgG utilized in the experiment was balanced with the 3D6 effector function (i.e., IgG2b); experiments performed with control IgG1 or IgG2a result in very similar values (data not shown). Primary murine microglia (8 × 105 cells, 500 μl) were then added to sections and incubated for 24 hr at 37°C. Each section with antibody treatment was followed by an untreated sister section. At the end of incubation, media were removed and tissue sections and cells were homogenized with 5.2 M guanidine buffer (300 μl), diluted 10× and 100× with PBS buffer containing 0.5 M guanidine, 0.05% Tween20 and 0.25% casein, and Aβ1-42 concentration quantified by ELISA. To account for differences in the amount of deposited Aβ in different sections, we normalized each unknown treatment by the untreated sister section. Data were directly plotted in GraphPad Prism and analyzed by one-way ANOVA with Newman-Keuls posttest.