Imaging autophagy.
Imaging autophagy.
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成像自噬。
DOI：
10.1002/0471142956.cy1234s69
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发表时间：
2014
影响因子：
--
通讯作者：
Karanasios E
中科院分区：
文献类型：
--
作者：
Karanasios E
自噬是一种膜运输途径，被激活以通过一种新的膜隔室（自噬体）将细胞溶质物质递送至溶酶体进行降解。荧光显微镜是用于可视化细胞内蛋白质的最常用方法，它被广泛用于自噬领域。为了将其与细胞背景区分开，将感兴趣的蛋白质（POI）与遗传编码的荧光蛋白融合或用与无机荧光化合物缀合的抗体染色。POI的遗传标记允许其在活细胞中可视化，而POI的免疫染色需要细胞的固定和细胞膜的透化。在这里，我们描述了详细的协议，如何可视化的自噬动态使用荧光显微镜在活细胞和固定的细胞。我们讨论了每种技术的关键参数，它们的优点，以及为什么当它们串联使用时，鲁棒性会增加。方案Cytom 69：12.34.1 - 12.34.16。© 2014年由John Wiley & Sons，Inc.
Autophagy is a membrane‐trafficking pathway activated to deliver cytosolic material for degradation to lysosomes through a novel membrane compartment, the autophagosome. Fluorescence microscopy is the most common method used to visualize proteins inside cells, and it is widely used in the autophagy field. To distinguish it from the cellular background, the protein of interest (POI) is either fused with a genetically encoded fluorescent protein or stained with an antibody that is conjugated to an inorganic fluorescent compound. Genetic tagging of the POI allows its visualization in live cells, while immunostaining of the POI requires the fixation of cells and the permeabilization of cell membranes. Here we describe detailed protocols on how to visualize autophagy dynamics using fluorescence microscopy in live and fixed cells. We discuss the critical parameters of each technique, their advantages, and why the robustness is increased when they are used in tandem.Curr. Protoc. Cytom. 69:12.34.1‐12.34.16. © 2014 by John Wiley & Sons, Inc.