Aβ(M1-42) fibrils [1.5 μM Aβ(M1-42) monomer equivalents] were incubated with BSA (1 μM; New England Biolabs) in 20 mM phosphate buffer (pH 8.0) (10 min, RT). Clusterin (0.15 μM) was added to the solution and further incubated (10 min, RT). The fibril sample was centrifuged (15,000 rpm, 20 min, 4°C), and the supernatant was removed. The pellet was resuspended in PBS containing 0.01% (v/v) Triton and 0.01% (v/v) Tween 20 (15 μl), and then the sample was centrifuged (15,000 rpm, 20 min, 4°C). The pellet was resuspended in PBS (10 μl). For the sonicated samples, Aβ(M1-42) fibrils (1.5 μM) were sonicated using a probe sonicator (Bandelin, SONOPULS HD 2070) for 1 min with 10% maximum power and 30% cycles before incubation with BSA and clusterin. The prepared fibril samples (5 μl) were applied to a carbon support film, 400 mesh, 3-mm nickel grid (EM Resolutions Ltd., Saffron Walden, UK) and incubated (5 min, RT). The grid was blocked with BSA (1 mg/ml) in PBS for 15 min and incubated with 1:100 G7 mouse anti-human clusterin monoclonal antibody [stock solution (2 mg/ml)] in PBS for 30 min. For the secondary antibody–only negative controls, samples were incubated with PBS only at this step. The grid was washed (three times for 5 min), first, in PBS/0.01% Triton/0.01% Tween 20 and then twice with PBS only, followed by incubation with 1:500 gold-labeled anti-mouse secondary antibody (BBI Solutions, Cardiff, UK) in PBS for 30 min. Last, the grid was washed three times as described above, twice with water, and then incubated for 2 min with 2% uranyl acetate (w/v). To remove excess uranyl acetate, the grid was washed twice with water and dried completely before imaging. The fibrils were imaged on a FEI Tecnai G2 TEM (Cambridge Advanced Imaging Centre, University of Cambridge, UK). Images were analyzed using the SIS MegaView II Image Capture System (Olympus, Tokyo, Japan).

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