Name: plunge freezing: a tool for the ultrastructural and immunolocalization studies of suspension cells in transmission electron microscopy
Uploaded: 2017-05-05T12:30:00
Description: Watch this Scientific Journal Video about Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy at JoVE.com

我们对此表示感谢M. Bouchecareilh，E.Tétaud，S. Duvezin-Caubet和A.德文的帮助和意见的手稿。我们非常感谢何地拍摄的图像波尔多成像中心的电子成像极。这项工作是由中心法国国家科学研究的支持。

1.福尔瓦网格薄膜的制备注意：在使用个人防护设备（手套，实验室外套，眼镜）在通风橱下执行氯仿操纵。使用400个目电子显微镜铜网格。与其他类型的网格（其它的网孔尺寸，金和镍网格），冷冻的质量较差。 <ol><li>制备0.3％的聚乙烯醇缩甲醛的氯仿100mL的溶液。允许不搅拌溶解过夜。 </li><li>把400目在玻璃小瓶中电子microscopycopper网格（直径3.05毫米）（高度为3厘米，直?...

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TEM是细胞器，细胞，和组织的超微结构观测的有效方法。冷冻固定/冷冻取代是目前两种超微结构和蛋白的抗原性的保存的最佳方法。化学固定剂渗透和行动非常缓慢，从而使结构重组的超微结构2的完全稳定之前。相反，冷冻固定/冷冻置换瞬间稳定蜂窝结构4。然而，冷冻固定/冷冻替代技术包括了许多限制和困难从而限制了它们的应用范围的。这涉及样品，冷?...

样品制备为任何电子显微镜研究成功的关键。常规EM固定是用于固定的组织或细胞进行透射电子显微镜（TEM）1的主要方法。首先，醛和四氧化锇用于化学固定在室温下该材料。然后，将材料脱水用有机溶剂，渗透，并包埋在环氧树脂中。此方法取决于固定剂的渗透速率进入细胞。因此，细胞内容物中的工件和提取通常被观察到2个。 冷冻固定显然是细胞结构3保存一个更好的选择，让他们完好。可以使用冷冻固定/冷冻置换方法来获得在薄树脂切片TEM图像4的最高质量。这种技术的目的是为了获得玻璃化的双ological样品没有冰晶形成或含有冰晶小到足以不损伤细胞的超微结构。高压冷冻（HPF）和超快速冷冻固定，其也被称为切入冷冻（PF），两种方法来cryofix样品。 HPF在单元固定不动的分子瞬时且避免了由常规EM固定的损害。几种类型的冷冻机和自动替换设备已经开发5。冷冻机器和消耗品（液氮，标本的载体，等等）是昂贵的，但它们允许生产高品质的电子显微照片6,7。 PF是已在50年代初采用和在文献中简单和便宜8 .During的PF程序，将制备的样品以很快的速度冷冻，得到冰晶尺寸小于3至5nm，描述了一种技术。为此，所述样品是陷入液态制冷剂，如乙烷，丙烷，或乙烷 - 丙烷混合物。自50年代以来，PF的改进已经完成，以使这项技术提供给用户更大的数字。高压冷冻目前冻结大量的各种样品的厚度大于50微米（最多为盘形样品的厚度为200微米）5的唯一可行的办法，而PF被广泛地用于图像的小物体（&lt;100nm的），如悬浮于无定形冰9的薄膜大分子复合物。较大的样品，例如真核细胞，可以通过PF cryofixed，但需要试样保持器，如毛细管铜管或三明治系统8，10，11。 这里，快速，易于使用和可用于各种悬浮细胞培养物的低成本暴跌冷冻/冻干替代技术被呈现。

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</table>

plunge freezing: a tool for the ultrastructural and immunolocalization studies of suspension cells in transmission electron microscopy

透射电子显微镜（TEM）是研究细胞超微结构，以本地化的蛋白质和以非常高的分辨率可视化大分子复合物不平凡的工具。但是，要获得尽可能接近原生状态，需要完善的样品保存。与醛常规电子显微镜（EM）固定，比如，不能提供良好的超微结构保存。固定剂的缓慢渗透诱导细胞重组和各种细胞成分的损失。因此，常规EM固定不允许结构和抗原性的瞬时稳定和保存。审查细胞内事件的最佳选择是使用冷冻固定，然后，保持细胞的天然状态的冷冻替代固定方法。高压冷冻/冻干取代，其保留细胞的超微结构的完整性，是最常用的方法，但需要expensi已经装备。这里，易于使用和低成本的冷冻固定方法进行冷冻取代悬浮细胞培养物呈现。

在本文中，提出了一种用于超微结构（ 图1和图2）和免疫标记（ 图3）的研究一种易于使用和低成本的暴跌冷冻方法。我们证明，这是没有必要有专用设备冷冻置换程序，该程序升温只需不到6个小时，而不是使用专用系统的24个小时。 PF /冷冻置换方法导致在<...

Watch this Scientific Journal Video about Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy at JoVE.com

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

此手稿描述了通过透射电子显微镜可视化用混悬剂细胞中易于使用和低成本的冷冻固定方法。

暴跌冻结：在透射电子显微镜的超微结构和免疫组化研究工具悬架细胞

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

Research

JoVE Journal

Biology

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

科研

生物学

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

在早期鸡胚跟踪形态组织变形

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

透射电镜的小型化样品制备方法

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

使用新型机器人系统 Spotiton 进行时间解决研究的低温电子微观网格准备

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

使用夹层冷冻装置快速冷冻，在电子显微镜下对生物标本进行良好的超微结构保存

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ...

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

原位 使用透射电子显微镜探索小鼠巨核细胞生成

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These ...