Electron microscopy of endocytic pathways.
Electron microscopy of endocytic pathways.
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内吞途径的电子显微镜。
作者：
C. Ranftler;Peter Auinger;C. Meisslitzer;A. Ellinger;J. Neumüller;M. Pavelka
详细了解内吞系统复杂动态区室的精细结构和 3D 架构对于从细胞表面到不同细胞内目的地的逆行交通的形态功能分析至关重要。在这里，我们描述了一种细胞化学方法，用于电子显微镜探索内吞途径，使用小麦胚芽凝集素（WGA）结合传统化学固定或通过高压冷冻对细胞进行超快物理固定。人肝癌细胞内吞不同时间段的辣根过氧化物酶标记的 WGA 作为标记。其细胞内途径通过二氨基联苯胺氧化可视化，该氧化可以在化学固定后常规进行，也可以在物理固定前在活细胞中进行。后一种方案允许将过氧化物酶催化的细胞化学与高压冷冻（HPF）相结合，这是在高空间和时间分辨率下对复杂和动态细胞器进行超微结构研究的最先进技术。该技术产生独特的细胞化学反应和保存完好的精细结构，非常适合复杂内吞结构的详细电子显微镜和 3D 研究。
Detailed insight into the fine structure and 3D-architecture of the complex and dynamic compartments of the endocytic system is essential for a morpho-functional analysis of retrograde traffic from the cell surface to different intracellular destinations. Here, we describe a cytochemical approach for electron microscopic exploration of endocytic pathways with the use of wheat germ agglutinin (WGA) in combination with either conventional chemical fixation or ultrafast physical fixation of the cells by high pressure-freezing. Horseradish peroxidase-labeled WGA endocytozed by human hepatoma cells for various periods of time served as a marker. Its intracellular routes were visualized by means of diaminobenzidine oxidation either done conventionally after chemical fixation or in living cells prior to physical fixation. The latter protocol permits the combination of peroxidase-catalyzed cytochemistry with high pressure-freezing (HPF), which is state of the art for ultrastructural studies of complex and dynamic organelles at high spatial and temporal resolutions. The technique yields distinct cytochemical reactions and excellently preserved fine structures well qualified for detailed electron microscopic and 3D-studies of the complex endocytic architectures.