A method for efficient observation of intracellular membranes of monolayer culture cells by quick-freeze and freeze-fracture electron microscopy.
A method for efficient observation of intracellular membranes of monolayer culture cells by quick-freeze and freeze-fracture electron microscopy.
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一种通过速冻和冷冻断裂电子显微镜有效观察单层培养细胞细胞内膜的方法。
DOI：
10.1093/jmicro/dfs063
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Hitomi Suzuki
中科院分区：
文献类型：
--
作者：
Akikazu Fujita;Toyoshi Fujimoto;Nami Ozato-Sakurai;Hitomi Suzuki
我们以前开发了一种方法，分析二维分布的膜脂结合快速冷冻和冷冻断裂复制标记（QF-FRL）。原则上，这种方法可以应用于任何膜，但在实践中，不容易在没有冰晶破坏的情况下有效地观察细胞质细胞器。在本文中，我们报告了我们的方法，绕过这个问题的修改。在改进的方法中，将细胞培养在用砂纸刮擦的金箔上，并根据高压冷冻法快速冷冻。该技术能够有效观察细胞内结构，如细胞核、内质网、高尔基体和线粒体。在所获得的冷冻断裂复制品中确认了磷酸肌醇4，5-二磷酸的特异性标记。目前的QF-FRL方法是易于使用，并应加快细胞内脂质分布的分析。
We previously developed a method of analyzing the two-dimensional distribution of membrane lipids by combining quick-freezing and freeze-fracture replica labeling (QF–FRL). In principle, this method can be applied to any membrane, but in practice it is not easy to observe cytoplasmic organelles efficiently without ice crystal damage. In this paper, we report a modification of our method that circumvents this problem. In the modified method, cells are cultured on a gold foil scratched with sandpaper and quick-frozen according to a high-pressure freezing method. This technique enables the efficient observation of intracellular structures such as the nucleus, endoplasmic reticulum, Golgi apparatus and mitochondria. Specific labeling of phosphoinositide 4,5-bisphosphate was confirmed in the obtained freeze-fracture replica. The present QF–FRL method is easy to use and should expedite the analysis of intracellular lipid distribution.