| Immunogold Electron Microscopy of the Autophagosome Marker LC3
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Author:
Date:
2017-12-20
[Abstract] Even though autophagy was firstly observed by transmission electron microscopy already in the 1950s (reviewed in Eskelinen et al., 2011), nowadays this technique remains one of the most powerful systems to monitor autophagic processes. The autophagosome, an LC3-positive double membrane structures enclosing cellular materials, represents the key organelle in autophagy and its simple visualization and/or numeration allow to draw important conclusions about the autophagic flux. Therefore, the accurate identification of autophagosomes is crucial for a comprehensive and detailed dissection of autophagy. Here we present a simple protocol to identify autophagosomes by transmission electron microscopy coupled to immunogold labeling of LC3 starting from a relatively low cell number, which ...
[摘要] 尽管早在20世纪50年代就已经通过透射电子显微镜观察了自噬（在Eskelinen等人2011年的综述中），但是现在这种技术仍然是监测自噬过程的最强大的系统之一。 自噬体是包含细胞物质的LC3阳性双层膜结构，代表了自噬的关键细胞器，其简单的可视化和/或计数允许得出关于自噬流的重要结论。 因此，准确鉴定自噬体对自噬的全面和详细的分析至关重要。 在这里我们提出一个简单的协议，以确定autophagosomes透射电子显微镜耦合LC3的免疫金标记从一个相对较低的细胞数量，我们最近开发遵循病毒介导的人类癌变期间的自噬途径。
【背景】自噬体代表了macroautophagy的关键结构，这是一种细胞胞质成分的分解代谢系统。巨自噬（或简单地自吞噬）由吞噬细胞的形成引发，所述吞噬细胞能够自身扩张吞噬细胞器和蛋白质，所述蛋白质最终闭合在螯合成分周围形成被称为自噬体的细胞器。接下来，在成熟过程中，自噬体可以与溶酶体融合以形成自溶酶体，其中被捕获的物质被位于溶酶体限制性膜中的泵降解并再循环回（Glick et al。，2010）。
由于自噬功能障碍与各种人类疾病，病毒感染，神经退行性疾病，免疫功能和癌症有关（Schneider and ...
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| A Bioimaging Pipeline to Show Membrane Trafficking Regulators Localized to the Golgi Apparatus and Other Organelles in Plant Cells
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Author:
Date:
2015-09-05
[Abstract] The plant Golgi apparatus is composed of numerous stacks of cisterna, designated as cis, medial, and trans Golgi cisternae; these stacks move within the cytoplasm along the actin cytoskeleton. Cis cisternae receive secretory products from endoplasmic reticulum (ER) and they subsequently progress through the stack to the trans cisternae, where they are sorted to other destinations, including cell wall, plasma membrane (PM), vacuoles, and chloroplasts. In addition, the plant Golgi apparatus plays a role of glycosylating proteins as well as synthesizing cell wall polysaccharides, such as hemicelluloses and pectins. This protocol describes procedures for imaging fluorescently-tagged proteins localized to the plant Golgi apparatus of Arabidopsis ...
[摘要] 植物高尔基体包括许多叠状体，称为顺，内侧和反式高尔基这些堆栈在细胞质内沿着肌动蛋白细胞骨架移动。 Ctern 池从内质网（ER）接收分泌产物，并且随后通过堆叠进入反转池，在那里它们被分选到其他目的地，包括细胞壁，血浆膜（PM），液泡和叶绿体。此外，植物高尔基体起到糖基化蛋白质以及合成细胞壁多糖如半纤维素和果胶的作用。该协议描述了使用共聚焦激光显微镜（CLSM），全内反射荧光显微镜（TIRF）和高压的免疫金标记标记定位于拟南芥幼苗的植物高尔基体的荧光标记的蛋白质的程序冷冻/冷冻取代的样品通过透射电子显微镜（TEM）。我们特别关注长期时间推移成像和蛋白质本地化在高尔基内的子域。该方案还可以用于其他细胞器，组织和植物物种。
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| Thin Sections of Technovit 7100 Resin of Rice Endosperm and Staining
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Author:
Date:
2014-09-20
[Abstract] Starch is a biologically and commercially important carbohydrate that is accumulated in plant storage organs, such as seed endosperm. Starch is water-insoluble and forms transparent grains, referred to as starch grains (SGs). SGs are easily stained by iodine solution and can be observed under a normal microscope. Technovit 7100 resin is suitable for preparation of thin sections from endosperm. The thin sections and iodine staining can visualize SGs clearly inside the endosperm cell. This protocol provides the procedures to prepare thin sections of Technovit 7100 resin from rice endosperm. It can also be applicable to the seeds of other plant species.
Figure 1. Iodine-stained ...
[摘要] 淀粉是在植物储存器官（例如种子胚乳）中积累的生物学上和商业上重要的碳水化合物。 淀粉是水不溶性的并且形成称为淀粉颗粒（SGs）的透明颗粒。 SGs容易被碘溶液染色，并且可以在正常显微镜下观察。 Technovit 7100树脂适用于从胚乳制备薄切片。 薄切片和碘染色可以明显地在胚乳细胞内部显现SG。 该协议提供了从水稻胚乳制备Technovit 7100树脂薄片的程序。 它也可以适用于其他植物物种的种子。
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