| Visualization of Cell Complexity in the Filamentous Cyanobacterium Mastigocladus laminosus by Transmission Electron Microscopy (TEM)
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Author:
Date:
2014-12-05
[Abstract] The cyanobacterium Mastigocladus laminosus (M. laminosus) is one of the most morphologically complex prokaryotes. It forms long chains of cells that are connected via septal junction complexes; such complexes allow diffusion of metabolites and regulators between neighboring cells. Cellular division occurs in multiple planes, resulting in the formation of true branches, and cell differentiation leads to the formation of specialized cell types for nitrogen fixation (heterocysts) and culture dispersal (hormogonia and necridia). Here, we describe a detailed protocol for the preparation of M. laminosus for TEM in order to visualize the ultrastructural properties of the organism. The presented preparation method is based on adding potassium permanganate as fixative ...
[摘要] 蓝藻（Mastigocladus laminosus）（ M。laminosus ）是最具形态复杂的原核生物之一。 它形成通过间隔连接复合物连接的细胞的长链; 这种复合物允许代谢物和调节剂在相邻细胞之间扩散。 细胞分裂发生在多个平面，导致真正的分支的形成，细胞分化导致固氮（heterocysts）和文化扩散（hormogonia和necridia）的专门细胞类型的形成。 在这里，我们描述了用于制备 M的详细方案。 层状结构用于TEM，以便可视化生物体的超微结构特性。 所提出的制备方法基于添加高锰酸钾作为固定剂，已显示增加膜的对比度（Luft，1956），使其适合于在其中光合膜的可视化是重要的蓝细菌的研究。
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| Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites
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Author:
Date:
2014-06-05
[Abstract] This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined using standard transmission electron microscopy (EM). Pre-embedding EM is a common method where the antibodies are introduced before embedding and sectioning. This method avoids the problem that antigens are often difficult to detect on EM-sections after embedding. In the method described here antigens in the parasite-infected host cell are detected. Entry of the antibody is made possible through permeabilisation of the host cell with tetanolysin. In principle this method could ...
[摘要] 该方案用于制备过表达用GFP标记的寄生虫蛋白PF13_0191的恶性疟原虫血液阶段寄生虫的预嵌入样品。使用GFP特异性抗体和蛋白A-Gold，使用标准透射电子显微镜（EM）测定过表达的蛋白在感染的宿主细胞中的定位。预嵌入EM是其中在包埋和切片之前引入抗体的常见方法。该方法避免了嵌入后在EM-切片上通常难以检测抗原的问题。在本文所述的方法中，检测寄生虫感染的宿主细胞中的抗原。通过宿主细胞用破伤风溶蛋白透化可以进入抗体。原则上，如果样品在添加相关抗体之前被适当地固定和透化，则该方法也可以用于检测寄生虫内的抗原。虽然抗体的获取将避免嵌入后方法中经常见到的检测问题，但是该方法将产生相对较差的形态。
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