Dot Blot Assay for Amyloid Fibrils

Dot blot assays were performed using the fibril-specific OC antibody [71 (link)]. Briefly, samples of 4 μL containing Aβ or α-syn were spotted onto a nitrocellulose membrane (Hybond-ECL, GE Life Sciences) and after air-drying, membranes were blocked with 2.5% BSA in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBS-T) for 1 h at room temperature. After rinsing briefly with TBS, membranes were probed with the OC antibody (1:2000 in TBS; AB2286, Millipore, Bedford, MA, USA) for 2 h at room temperature. The membranes were then washed three times for 5 min each with TBS-T on an orbital shaker, and incubated with secondary horseradish peroxidase-conjugated anti-rabbit antibody (1:5000 in TBST) for 1 h at room temperature. Three subsequent washes were performed with TBS-T and the last wash with TBS only, for 5 min each. Lastly, the blots were developed using the ECL immunoblotting kit (RPN2108, GE Life Sciences, Little Chalfont, United Kingdom) as per manufacturer instructions.