Fig. 1
From: Amyloid fibril proteomics of AD brains reveals modifiers of aggregation and toxicity
A purification strategy to isolate amyloid fibrils from AD and AD model brain extracts. a Biochemical purification strategy schematic. This method builds on previously developed methods and purifies SDS-insoluble dense amyloid fibril cores with sucrose density-gradient ultracentrifugation, ultrasonication, and washing with SDS. P = pellet, and S = supernatant. P6 = previously reported amyloid fibrils, P11 = highly purified amyloid fibrils. b Western blot analysis of indicated fractions collected during amyloid fibril purification from transgenic 5xFAD cortical extracts using anti-fibril (LOC) antibody. 10% v/v material from each fraction was loaded. c Normalized relative abundance of LOC-positive species in P11 with respect to input (brain homogenate). d Representative purified amyloid material (i.e., P11 fraction from AppNL−G−F/NL−G−F cortical extracts) stained with Congo red (CR) and visualized under cross polarized light. The image was captured using a monochromatic camera and is presented in greyscale. e Immunofluorescence (IF) images of P11 fractions from AppNL−G−F/NL−G−F mouse using Aβ42 and LOC antibodies. f Representative negative staining EM analysis of amyloid material (P11) extracted using our purification strategy from AppNL−G−F/NL−G−F brains compared to amyloids (P6) enriched using previously reported purification strategy. g Immunogold labeling with Aβ42 antibodies of purified fibrils, visualized by negative staining EM. IgG antibody was used as negative control. h, i WB analysis of purified fibrils isolated from cortical extracts of WT, 5xFAD, and App KI (AppNL/NL, AppNL−F/NL−F, and AppNL−G−F/NL−G−F) mouse lines with contrasting levels of amyloid pathology. j, k WB analysis and quantification of amyloid fibrils isolated from human brain tissues with increasing amyloid pathology (amyloid scores) with LOC antibody. Data in c and k represents mean ± SEM; *, p-value < .05; **, p-value < .01; ***, p-value < .001; analyzed with unpaired Student’s t-test or one-way ANOVA with post-hoc Sidek test. NL = AppNL/NL, NL-F = AppNL−F/NL−F, NL-G-F = App.NL−G−F/NL−G−F; P = pellet, and S = supernatant. N = 3 mice (d, e, and g), 5 mice (b, c), 6—8 mice (h, i), 5 mice (f); N = 3 (j and k). All mice were 6 months of age. Scale bar = 100 µm (d), 10 µm (e), 500 nm (f), 50 nm (g)