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

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 c...

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Detalles Bibliográficos
Autores principales: Bayati, Armin, Luo, Wen, Del Cid-Pellitero, Esther, Fon, Edward A., Durcan, Thomas M., McPherson, Peter S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9988663/
https://www.ncbi.nlm.nih.gov/pubmed/36861831
http://dx.doi.org/10.1016/j.xpro.2023.102113
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author Bayati, Armin
Luo, Wen
Del Cid-Pellitero, Esther
Fon, Edward A.
Durcan, Thomas M.
McPherson, Peter S.
author_facet Bayati, Armin
Luo, Wen
Del Cid-Pellitero, Esther
Fon, Edward A.
Durcan, Thomas M.
McPherson, Peter S.
author_sort Bayati, Armin
collection PubMed
description 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)
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spelling pubmed-99886632023-03-08 Visualization of α-synuclein trafficking via nanogold labeling and electron microscopy Bayati, Armin Luo, Wen Del Cid-Pellitero, Esther Fon, Edward A. Durcan, Thomas M. McPherson, Peter S. STAR Protoc Protocol 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) Elsevier 2023-02-24 /pmc/articles/PMC9988663/ /pubmed/36861831 http://dx.doi.org/10.1016/j.xpro.2023.102113 Text en © 2023 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Bayati, Armin
Luo, Wen
Del Cid-Pellitero, Esther
Fon, Edward A.
Durcan, Thomas M.
McPherson, Peter S.
Visualization of α-synuclein trafficking via nanogold labeling and electron microscopy
title Visualization of α-synuclein trafficking via nanogold labeling and electron microscopy
title_full Visualization of α-synuclein trafficking via nanogold labeling and electron microscopy
title_fullStr Visualization of α-synuclein trafficking via nanogold labeling and electron microscopy
title_full_unstemmed Visualization of α-synuclein trafficking via nanogold labeling and electron microscopy
title_short Visualization of α-synuclein trafficking via nanogold labeling and electron microscopy
title_sort visualization of α-synuclein trafficking via nanogold labeling and electron microscopy
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9988663/
https://www.ncbi.nlm.nih.gov/pubmed/36861831
http://dx.doi.org/10.1016/j.xpro.2023.102113
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