Multiplex Immunofluorescent Analysis of Alpha‐Synuclein in Nigral Lewy Bodies With Heat‐Induced Antibody Stripping Reveals an Intricate Multilayered Structure

In the study, we employ an affordable, tissue-saving, and precise simultaneous multiplex immunofluorescence method with heat-induced antibody stripping to identify and structurally analyse nigral Lewy bodies in dopaminergic neurones. Analysis of different alpha-synuclein epitopes and proteoforms reveals an almost uniform, onion-like morphology of the Lewy bodies. The N-terminal and C-terminal domains are predominantly accessible to antibody binding in the peripheral shell of the bodies.