Martinet Wim, Timmermans Jean-Pierre, De Meyer Guido R Y
Laboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium.
Laboratory of Cell Biology and Histology, University of Antwerp, Antwerp, Belgium.
Methods Enzymol. 2014;543:89-114. doi: 10.1016/B978-0-12-801329-8.00005-2.
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.
自噬是一种高度保守的溶酶体降解途径，在肿瘤发生和肿瘤进展中均起主要作用。透射电子显微镜（TEM）以及免疫组织化学是原位评估自噬不可或缺的工具。在此，我们描述一种通过TEM研究自噬泡的优化方案，并详细阐述微管相关蛋白1轻链（MAP1LC3，通常称为LC3）的免疫组织化学检测，LC3目前被认为是自噬过程最可靠的标志物之一。讨论了这些方法的优点、潜在陷阱和局限性，以及它们在自噬和肿瘤代谢研究领域的价值。总体而言，我们建议联合使用不同技术，包括TEM、免疫组织化学和分子方法（如免疫印迹），以明确检测恶性组织和正常组织中的自噬。
