在组织培养细胞中通过扫描电子显微镜对细胞核和核膜进行原位可视化观察。

Visualization of the nucleus and nuclear envelope in situ by SEM in tissue culture cells.

作者信息

Allen T D, Rutherford S A, Murray S, Gardiner F, Kiseleva E, Goldberg M W, Drummond S P

机构信息

Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Withington, Manchester M20 4BX, UK.

出版信息

Nat Protoc. 2007;2(5):1180-4. doi: 10.1038/nprot.2007.139.

DOI:10.1038/nprot.2007.139

PMID:17546013

Abstract

Our previous work characterizing the biogenesis and structural integrity of the nuclear envelope and nuclear pore complexes (NPCs) has been based on amphibian material but has recently progressed into the analysis of tissue-culture cells. This protocol describes methods for the high resolution visualization, by field-emission scanning electron microscopy (FESEM), of the nucleus and associated structures in tissue culture cells. Imaging by fluorescence light microscopy shows general nuclear and NPC information at a resolution of approximately 200 nm, in contrast to the 3-5 nm resolution provided by FESEM or transmission electron microscopy (TEM), which generates detail at the macromolecular level. The protocols described here are applicable to all tissue culture cell lines tested to date (HeLa, A6, DLD, XTC and NIH 3T3). The processed cells can be stored long term under vacuum. The protocol can be completed in 5 d, including 3 d for cell growth, 1 d for processing and 1 d for imaging.

摘要

我们之前关于核膜和核孔复合体（NPCs）生物发生及结构完整性的研究工作基于两栖类材料，但最近已发展到对组织培养细胞的分析。本方案描述了通过场发射扫描电子显微镜（FESEM）对组织培养细胞中的细胞核及相关结构进行高分辨率可视化的方法。荧光显微镜成像显示的是约200 nm分辨率下的一般细胞核和NPC信息，与之形成对比的是FESEM或透射电子显微镜（TEM）提供的3 - 5 nm分辨率，后者能在大分子水平上呈现细节。这里描述的方案适用于迄今测试过的所有组织培养细胞系（HeLa、A6、DLD、XTC和NIH 3T3）。处理后的细胞可在真空中长期保存。该方案可在5天内完成，包括细胞生长3天、处理1天和成像1天。