Fig. 5

Immunization with 43D but not 77E9 decreases Aβ pathology in 3×Tg-AD mice. a–f Analysis of Aβ pathology 1 week after seventh dose (see Fig. 1). a Representative Western blots of forebrain developed with APP. Densitometric quantification of blots after normalization with GAPDH. Immunostaining of (b) CA1 with 4G8 and (c) subiculum with thioflavin S in 3×Tg-AD mice. Quantification of APP staining with 4G8 in CA1 and amyloid plaques in subiculum stained with thioflavin S from two sections per mouse of five or six mice per group. The levels of total (d) Aβ₄₀ and (e) Aβ₄₂ (unpaired t test, 3×Tg-IgG vs. 3×Tg-43D, p = 0.08) and (f) the ratio of Aβ₄₂/Aβ₄₀ in forebrain was quantified by ELISA. g–i Aβ pathology in 3×Tg-AD mice 1 day after the sixth injection. 4G8 staining in (g) CA1 and (h) subiculum and (i) thioflavin S staining in subiculum in 3×Tg-AD mice. 43D did not cross-react with Aβ (j) and Aβ plaques (k). j Thirty micrograms of 3×Tg-AD mouse brain homogenate (lanes 1–3) and 0.4 μg of purified Aβ (lane 4) were loaded. Nitrocellulose membrane was first developed with 43D antibody (left panel) and then developed with 4G8 antibody (right panel). k Double-stained 3×Tg-AD mouse brain sections with 43D antibody and thioflavin S. Top row: CA1 and subiculum area (original magnification × 10). Bottom row: Subiculum area (original magnification × 20). Quantification of APP staining in CA1 and amyloid plaques in subiculum stained with 4G8 and thioflavin S from two sections per mouse of three or five animals per group. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by ANOVA followed by a Bonferroni post hoc test for two doses of immunization or by unpaired two-tailed t test for six doses of immunization. AD Alzheimer’s disease, ANOVA Analysis of variance, APP Amyloid precursor protein, Aβ Amyloid-β, ELISA Enzyme-linked immunosorbent assay, GAPDH Glyceraldehyde 3-phosphate dehydrogenase, IgG Immunoglobulin G, MW Molecular weight, 3×Tg Triple-transgenic, TS Thioflavin S, WT Wild type