Improved procedures for electron microscopic visualization of the cytoskeleton of cultured cells
Improved procedures for electron microscopic visualization of the cytoskeleton of cultured cells
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DOI：
10.1006/jsbi.1995.1054
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发表时间：
1995-11-01
影响因子：
3
通讯作者：
Borisy, GG
中科院分区：
文献类型：
--
作者：
Svitkina, TM;Verkhovsky, AB;Borisy, GG
我们开发了一种改进的电子显微镜方法，适用于细胞骨架的相关光和电子显微镜。该程序基于洗涤剂提取、化学固定、临界点干燥和培养细胞的铂/碳涂层，以及由个别微小但共同产生重大影响的修改组成的改进。它们是：在提取介质中加入聚乙二醇；在室温下裂解细胞；通过顺序应用戊二醛、单宁酸和醋酸铀酰进行固定；脱水和干燥过程中样品的水平位置；以及脱水过程中醋酸铀酰的处理。结果，我们获得了极大地提高了电子显微镜图像的质量以及结果的高度一致性。肌动蛋白细丝在应力纤维和新形成的片状脂肪中清晰可见。用肌球蛋白亚片段1修饰它们的极性明显。通过明胶处理细胞骨架上的肌动蛋白可以更好地显示肌球蛋白、中间丝和微管。通过这种处理暴露出来的中间细丝显示出许多侧面突起，呈现出迄今未见报道的千足虫样外观。所建议的程序与免疫金标记法兼容，如微管蛋白抗体所示。显微注射肌球蛋白II的荧光衍生物的细胞的相关光学和电子显微镜是可靠和有效的，产生了两种图像的相似之处。(C)1995年学术出版社。
We have developed an improved electron microscopic procedure appropriate for correlative light and electron microscopy of the cytoskeleton. The procedure is based on detergent extraction, chemical fixation, critical point drying, and platinum/carbon coating of cultured cells and the improvements consistof modifications which are minor individually but collectively of substantial impact. They are: inclusion of polyethylene glycol into the extraction medium; cell lysis at room temperature; fixation by sequential application of glutaraldehyde, tannic acid, and uranyl acetate; horizontal position of specimens during dehydration and drying; and uranyl acetate treatment during dehydration. As a result, we have obtained a greatly improved quality of electron microscopic images together with a high consistency of results. Long and straight actin filaments were clearly seen in stress fibers and newly formed lamellipodia. Their polarity was distinctly revealed by decoration with myosin subfragment 1. Depletion of actin from cytoskeletons by gelsolin treatment allowed for better visualization of myosin, intermediate filaments, and microtubules. Intermediate filaments exposed by this treatment exhibited numerous side projections in a hitherto unreported millipede-like appearance. The suggested procedure was compatible with immunogold labeling as demonstrated with an antibody to tubulin. Correlative light and electron microscopy of cells microinjected with a fluorescent derivative of myosin II was reliable and efficient, producing a close resemblance between the two kinds of images. (C) 1995 Academic Press, Inc.