Spatial mapping of α-synuclein seeding activity and pathology using immunofluorescence-integrated quiescent seed amplification

Seed amplification assays sensitively detect misfolded alpha-synuclein (alphaSyn) templating activity in homogenates but erase tissue coordinates, whereas Ser129-phosphorylated alphaSyn (pS129) immunostaining preserves anatomy without measuring functional seeding competence. Here we report multiplexed immunofluorescence-integrated quiescent seed amplification (IF-QSAA), coupling in situ QSAA with multichannel immunolabelling on the same brain section. Combining mouse alphaSyn monomer, high sulfate and 70 degrees C quiescent incubation under an agarose overlay generates localized, fibrillar thioflavin T-positive products with high seed specificity. Across preformed-fibril-inoculated mouse brains and human Parkinson's disease (PD) tissue, QSAA reveals white-matter seeding enrichment, resolves hemispheric asymmetry earlier than pS129, and recovers endogenous seeding signals from archival formalin-fixed PD tissue lacking pS129 immunoreactivity. Using a QSAA-first workflow with 80 degrees C citrate retrieval and spectral unmixing, IF-QSAA identifies selective neuronal and microglial associations and resolves core-halo QSAA-pS129 dual-positive pairs. IF-QSAA thus co-maps functional seeding and molecular histopathology.