Fig. 2: Validation of the specificity of the α-syn-isSID assay.

Overview of the α-syn-isSID assay setup. A marked increase in the ThT fluorescence was detected exclusively in cases with previously characterized α-syn pathology, exemplified by a representative PD case (green box). No relative increase in ThT signal was detected in a representative control case or in the technical negative where His-α-syn monomer was not added (red boxes) (a). Areas of positive α-syn-isSID signal corresponded with areas of α-syn pathology detected by IHC in PD cases. Using amygdala sections from the same patient, α-syn-isSID and α-syn-IHC were performed, where a representative case with high pathology (top) and low pathology (bottom) are shown (b). In the amygdala of a PD case, extensive seeding-competent α-syn pathology was observed (gray square), while neurons lacking α-syn pathology (blue square) were also present, confirming signal specificity. Omission of either the anti-His antibody or recombinant His-α-syn substrate during incubation abolished signal, confirming that the assay specifically detects newly aggregated monomeric His-α-syn in situ (c). Control cases without α-syn pathology, as confirmed by α-syn-IHC and AS-PLA, showed no signal in α-syn-isSID (d). RT-QuIC curves confirm consistency with α-syn-isSID results. Positive RT-QuIC responses were observed in PD, DLB, and MSA cases with α-syn-isSID signal, while control cases lacking both α-syn pathology and α-syn-isSID signal showed negative RT-QuIC responses. RT-QuIC traces represent averaged triplicate samples, and fluorescence is shown in relative fluorescence units (RFU) (e). Scale bar is 100 µm; insets show magnified regions.