Fig. 1

Characterization of anti-α-syn antibodies. 50 ng of monomers (M) and fibrils (F) of human α-syn, ABri, Islet amyloid polypeptide (IAPP), Abeta or tau (a), and β- or γ-syn (b) were spotted onto nitrocellulose membranes, and then probed with the indicated antibodies. c Representative photomicrographs illustrating histopathological features detected with Syn-O2 in substantia nigra pars compacta (SNpc), CA2 and deep layers of entorhinal cortex (dENT) or superficial layers of entorhinal cortex (sENT) of a PD patient (male, 84 years; Braak PD stage 6). 1) Lewy body and small bulgy neurites in SNpc; 2) neuron with punctated Syn-O2 immunoreactivity in SNpc. *Note the Syn-O2 immunoreactive beaded strings. 3) long thin threads and many small extracellular or synaptic Syn-O2 immunoreactivity; 4) a long thread in CA2 and many small neurites; 5) Lewy body pathology in deep layers of enthorinal cortex; 6) many small neurites and synaptic staining (Bar = 50 μm). d Photomicrographs showing immunoreactivity of Syn-O2, KM-51 and Syn-1 monoclonal antibodies in 10 μm thick paraffin-embedded sections of a control and PD patient. No staining was observed in the control with any of these mAbs. In PD patient, small and large LBs and LNs were observed with all three mAbs. With Syn-O2, many other long neurites and small extracellular or synaptic-like aggregates were stained. Bar = 50 μm. e Monomeric (m-α-syn), oligomeric (o-α-syn), phosphorylated α-syn (p-Ser129-α-syn) and nitrated (n-α-syn) forms were used to test the cross-reactivity of our antibodies 11D12 and Syn-140 · The generic commercial mAbs Syn-1 (BD Biosciences, 50 ng/ml) for α-syn, and EP1536Y (Abcam) for p-Ser129-α-syn, were also included as controls. f 50 ng of recombinant α-, β- and γ-syn were loaded on SDS gels and transferred to nitrocellulose membranes for western blotting, and then probed with our antibodies or control antibodies as appropriate. Syn-1 (BD Biosciences, 50 ng/ml) for α-syn, anti-β-synuclein (8) (Santa Cruz Biotechnology, 1:2 K) for β-syn and C-20 (Santa Cruz Biotechnology, 1:3 K) for γ-syn. g 15 μg of human, mouse and rat brain lysates were used in western blotting to test the specificity of 11D12 and Syn-140 · 50 ng of recombinant human α-syn (rec.α-syn) was included as positive control. h 50 ng of recombinant human α-syn (H-α-syn) or mouse α-syn (M-α-syn), human phosphorylated α-syn (H-p-Ser129-α-syn) and mouse phosphorylated α-syn (M-p-S129-α-syn) were loaded on SDS gels and transferred to nitrocellulose membranes for western blotting, and the membranes were then probed with our mouse mAb (PS129) recognizes both human and mouse p-Ser129-α-syn or the generic commercial mAb Syn-1 as indicated