Visualization of α-synuclein trafficking via nanogold labeling and electron microscopy

Armin Bayati; Wen Luo; Esther Del Cid-Pellitero; Edward A Fon; Thomas M Durcan; Peter S McPherson

doi:10.1016/j.xpro.2023.102113

Visualization of α-synuclein trafficking via nanogold labeling and electron microscopy

STAR Protoc. 2023 Mar 17;4(1):102113. doi: 10.1016/j.xpro.2023.102113. Epub 2023 Feb 24.

Authors

Armin Bayati¹, Wen Luo², Esther Del Cid-Pellitero³, Edward A Fon³, Thomas M Durcan⁴, Peter S McPherson⁵

Affiliations

¹ Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC H3A 2B4, Canada. Electronic address: armin.bayati@mail.mcgill.ca.
² The Neuro's Early Drug Discovery Unit (EDDU), McGill University, Montreal, QC H3A 2B4, Canada.
³ Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC H3A 2B4, Canada.
⁴ The Neuro's Early Drug Discovery Unit (EDDU), McGill University, Montreal, QC H3A 2B4, Canada. Electronic address: thomas.durcan@mcgill.ca.
⁵ Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC H3A 2B4, Canada. Electronic address: peter.mcpherson@mcgill.ca.

Abstract

There is conflicting evidence regarding the mechanisms of α-synuclein internalization, and its trafficking itinerary following cellular entry remains largely unknown. To examine these issues, we describe steps for coupling α-synuclein preformed fibrils (PFFs) to nanogold beads and their subsequent characterization by electron microscopy (EM). Then we describe the uptake of conjugated PFFs by U2OS cells plated on Permanox 8-well chamber slides. This process eliminates the reliance on antibody specificity and the need to employ complex immunoEM staining protocols. For complete details on the use and execution of this protocol, please refer to Bayati et al. (2022).¹.

Keywords: Cell culture; Microscopy; Molecular/Chemical Probes; Neuroscience.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cells, Cultured
Microscopy, Electron
Neurons*
alpha-Synuclein*

Substances

alpha-Synuclein