Mouse anti alpha synuclein 211

Ten micrograms of protein from cell lysates or 0.1 μg of recombinant protein was suspended in Laemmli buffer and heated to 95°C for 10 min for SDS-PAGE, or suspended in SDS-free sample buffer for non-denaturing PAGE. Proteins were separated on 10% SDS-polyacrylamide or non-denaturing polyacrylamide gels, except in Fig. 3A where proteins were separated on a pre-cast Bio-Rad Criterion™ TGX™ 4–15% gradient gel. After transfer of proteins to polyvinylidene difluoride (PVDF) membranes (Millipore) and blocking in 3% (w/v) powdered skimmed milk in Tris-buffered saline/0.1% Tween 20 (TBS-T), membranes were incubated overnight at 4°C in primary antibody [mouse anti-alpha synuclein 211 (Abcam) or mouse anti-amyloid β 4G8 (Covance)] diluted in blocking solution. After washing three times in TBS-T, the membrane was incubated with secondary antibody for 1 h at room temperature. The membrane was washed again in TBS-T and the signal visualized with ECL reagent (Millipore) and exposure in the Bio-Rad Gel Doc™.

Paraffin embedded tissue was dewaxed in xylene and rehydrated in graded alcohols then blocked in 10% H₂O₂ for 1 h at room temperature in the dark to quench endogenous peroxidases. Antigens were retrieved by microwave heating with citrate buffer, pH 6, for a total of 10 min. Tissue was then blocked in 10% normal goat serum in TBS + 0.1% Triton™ X-100 for 1 h at room temperature. Primary antibodies (mouse anti-alpha-synuclein 211, Abcam; 1:2000) were incubated with the tissue overnight at 4°C, followed by washing and incubation with biotinylated goat anti-mouse IgG secondary antibody (Jackson Immunoresearch) for 1 h at room temperature. After washing, VectaStain ABC reagent (Vector Labs) was added for 1 h at room temperature, then sections were incubated with 3,3′-Diaminobenzidine (DAB, Sigma) substrate for 3 min. The tissue sections were then counterstained with haematoxylin and dehydrated in graded alcohols and xylene, before mounting with DPX mounting reagent.