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Related Concept Videos

Immunogold Electron Microscopy01:20

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Related Experiment Video

Updated: Sep 27, 2025

Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy
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An optimized protocol for immuno-electron microscopy of endogenous LC3.

Ann De Mazière1, Jan van der Beek1, Suzanne van Dijk1

  • 1Section Cell Biology, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.

Autophagy
|April 7, 2022
PubMed
Summary

This study optimizes immunoelectron microscopy for microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3), a key autophagy marker. The developed method allows ultrastructural localization of endogenous LC3 in cells and tissues without permeabilization.

Keywords:
AutophagyCLEMLC3bafilomycin A1immuno-electron microscopyultrathin cryosections

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Area of Science:

  • Cell Biology
  • Autophagy Research
  • Microscopy Techniques

Background:

  • Microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3) is a crucial marker for autophagic compartments.
  • Ultrastructural identification of LC3-labeled compartments via immuno-electron microscopy (immuno-EM) is essential but technically challenging.
  • Existing methods for endogenous LC3 immuno-EM are difficult and often require cell permeabilization.

Purpose of the Study:

  • To develop and optimize an immuno-EM protocol for reliable detection of endogenous LC3.
  • To characterize the localization of LC3 in autophagic compartments at ultrastructural resolution.
  • To provide a robust tool for studying LC3's role in autophagy under native conditions.

Main Methods:

  • Screening of commercially available antibodies for LC3 immuno-EM.
  • Application of optimized labeling conditions using ultrathin cryosections and protein A-gold conjugates.
  • Utilizing on-section correlative light-electron microscopy (CLEM) for precise localization.
  • Simultaneous labeling of LC3 with other markers like LAMP1 and SQSTM1/p62.

Main Results:

  • An optimized LC3 immuno-EM protocol was established, enabling detection without cell permeabilization.
  • Endogenous LC3 was localized to various stages of autophagic compartments, including early and late endosomes and autolysosomes.
  • Bafilomycin A1 treatment was shown to lead to LC3 accumulation within autolysosomes, not inhibit autophagosome-lysosome fusion.
  • The method proved robust for simultaneous detection of multiple autophagy-related proteins.

Conclusions:

  • This study presents the first comprehensive overview of endogenous LC3 localization at ultrastructural resolution using a commercially available antibody.
  • The optimized protocol offers a valuable tool for researchers investigating the canonical and non-canonical functions of LC3 in native cellular environments.
  • The findings clarify the impact of bafilomycin A1 on the autophagy pathway, specifically regarding autolysosome content.