An optimized protocol for immuno-electron microscopy of endogenous LC3

Ann De Mazière, Jan van der Beek, Suzanne van Dijk, Cecilia de Heus, Fulvio Reggiori, Masato Koike, Judith Klumperman*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

5 Citations (Scopus)
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MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) is widely used as marker of autophagic compartments at different stages of maturation. Electron microscopy (EM) combined with immunolabeling is the only technique that can reveal the ultrastructural identity of LC3-labeled compartments. However, immuno-EM of endogenous LC3 proteins has proven difficult. Here, we test a panel of commercially available antibodies and apply different labeling conditions to present an optimized procedure for LC3 immuno-EM. Using ultrathin cryosections and protein A-colloidal gold or gold enhancement labeling, we localize endogenous LC3 in starved cells or tissues in the presence or absence of the proton pump inhibitor bafilomycin A1. We localize LC3 to early and late stage autophagic compartments that can be classified by their morphology. By on-section correlative light-electron microscopy (CLEM) we show that comparable fluorescent LC3-positive puncta can represent different autophagic intermediates. We also show that our approach is sufficiently robust to label endogenous LC3 simultaneously with other lysosomal and autophagy markers, LAMP1 or SQSTM1/p62, and can be used for quantitative approaches. Thus, we demonstrate that bafilomycin A1 treatment from 2.5 up to 24 h does not inhibit fusion between autophagosomes and lysosomes, but leads to the accumulation of LC3-positive material inside autolysosomes. Together, this is the first study presenting an extensive overview of endogenous LC3 localization at ultrastructural resolution without the need for cell permeabilization and using a commercially available antibody. This provides researchers with a tool to study canonical and non-canonical roles of LC3 in native conditions.

Original languageEnglish
Pages (from-to)3004-3022
Number of pages19
Issue number12
Early online date7-Apr-2022
Publication statusPublished - 2022

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