title: Methods to assess autophagy in situ--transmission electron microscopy versus immunohistochemistry.
abstract: Autophagy is a well-conserved lysosomal degradation pathway that plays a major role in both oncogenesis and tumor progression. Transmission electron microscopy (TEM) as well as immunohistochemistry are indispensable tools for the evaluation of autophagy in situ. Here, we describe an optimized protocol for the study of autophagic vacuoles by TEM and elaborate on the immunohistochemical detection of microtubule-associated protein 1 light chain (MAP1LC3, best known as LC3), which is currently considered as one of the most reliable markers of the autophagic process. The advantages, potential pitfalls, and limitations of these methods, as well as their value in the field of autophagy and oncometabolism research are discussed. Overall, we recommend a combined use of different techniques including TEM, immunohistochemistry, and molecular approaches (such as immunoblotting) for the unambiguous detection of autophagy in malignant as well as in normal tissues.
authors.affiliation: Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium. Electronic address: wim.martinet@uantwerpen.be.
authors.forename: Wim
authors.initials: W
authors.lastname: Martinet
authors.affiliation: Laboratory of Cell Biology and Histology, University of Antwerp, Antwerp, Belgium.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Timmermans
authors.affiliation: Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium.
authors.forename: Guido R Y
authors.initials: GR
authors.lastname: De Meyer
mesh_terms: D000818:Animals; D001343:Autophagy; D007150:Immunohistochemistry / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D011817:Rabbits; D014617:Vacuoles / Q000648:ultrastructure
keywords: Autophagic vacuole; Autophagy; Immunohistochemistry; LC3; Lysosome; Transmission electron microscopy