Shakes Diane C, Miller David M, Nonet Michael L
Department of Biology, College of William and Mary, Williamsburg, Virginia, USA.
Methods Cell Biol. 2012;107:35-66. doi: 10.1016/B978-0-12-394620-1.00002-3.
Immunofluorescence microscopy is a powerful technique that is widely used by researchers to assess both the localization and endogenous expression levels of their favorite proteins. The application of this approach to C. elegans, however, requires special methods to overcome the diffusion barrier of a dense, collagen-based outer cuticle. This chapter outlines several alternative fixation and permeabilization strategies for overcoming this problem and for producing robust immunohistochemical staining of both whole animals and freeze-fractured samples. In addition, we provide an accounting of widely used antibody reagents available to the research community. We also describe several approaches aimed at reducing non-specific background often associated with immunohistochemical studies. Finally, we discuss a variety of approaches to raise antisera directed against C. elegans antigens.
免疫荧光显微镜检查是一项强大的技术，研究人员广泛使用它来评估他们感兴趣的蛋白质的定位和内源性表达水平。然而，将这种方法应用于秀丽隐杆线虫需要特殊的方法来克服基于胶原蛋白的致密外皮的扩散屏障。本章概述了几种替代的固定和通透策略，以克服这个问题，并对完整动物和冷冻断裂样本进行强有力的免疫组织化学染色。此外，我们还介绍了研究界广泛使用的抗体试剂。我们还描述了几种旨在减少免疫组织化学研究中常见的非特异性背景的方法。最后，我们讨论了多种针对秀丽隐杆线虫抗原产生抗血清的方法。
