Allen T D, Rutherford S A, Murray S, Sanderson H S, Gardiner F, Kiseleva E, Goldberg M W, Drummond S P
Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Withington, Manchester M20 4BX, UK.
Nat Protoc. 2007;2(5):1166-72. doi: 10.1038/nprot.2007.137.
This protocol details methods for the isolation of oocyte nuclear envelopes (NEs) from the African clawed toad Xenopus laevis, immunogold labeling of component proteins and subsequent visualization by field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). This procedure involves the initial removal of the ovaries from mature female X. laevis, the dissection of individual oocytes, then the manual isolation of the giant nucleus and subsequent preparation for high-resolution visualization. Unlike light microscopy, and its derivative technologies, electron microscopy enables 3-5 nm resolution of nuclear structures, thereby giving unrivalled opportunities for investigation and immunological characterization in situ of nuclear structures and their structural associations. There are a number of stages where samples can be stored, although we recommend that this protocol take no longer than 2 d. Samples processed for FESEM can be stored for weeks under vacuum, allowing considerable time for image acquisition.
本方案详细介绍了从非洲爪蟾（非洲爪蟾）分离卵母细胞核膜（NE）、对组成蛋白进行免疫金标记以及随后通过场发射扫描电子显微镜（FESEM）和透射电子显微镜（TEM）进行可视化的方法。该程序包括首先从成熟雌性非洲爪蟾中取出卵巢，解剖单个卵母细胞，然后手动分离巨大细胞核并随后进行高分辨率可视化准备。与光学显微镜及其衍生技术不同，电子显微镜能够实现3 - 5纳米的核结构分辨率，从而为原位研究和免疫表征核结构及其结构关联提供了无与伦比的机会。虽然有多个阶段可以储存样品，但我们建议本方案的执行时间不超过2天。用于FESEM处理的样品可以在真空下储存数周，这为图像采集留出了相当长的时间。
