Boykins Lou G, Jones Jonathan C R, Estraño Carlos E, Schwartzbach Steven D, Skalli Omar
Department of Biological Sciences, The University of Memphis, 103 Life Sciences Bldg, Memphis, TN, 38152, USA.
School of Molecular Biosciences, Washington State University, BLS 202F, 1770 NE Stadium Way, Pullman, WA, 99164, USA.
Methods Mol Biol. 2016;1474:217-32. doi: 10.1007/978-1-4939-6352-2_13.
Localization of specific proteins within cells at the nanometer level of resolution is central to understanding how these proteins function in cell processes such as motility and intracellular trafficking. Such localization can be achieved by combining transmission electron microscopy (TEM) with immunogold labeling. Here we describe a pre-embedding, indirect gold immunolabeling approach to localize two different proteins of interest with secondary antibodies labeled with gold particles of different sizes in cells grown on cover slips. In this protocol, the cells are immunolabeled prior to being embedded in an epoxy resin for ultrathin sectioning. The protocol also includes strategies for optimizing the balance between ultrastructure and antigen preservation, steps to minimize nonspecific antibody binding, and steps to optimize antibody penetration.
在纳米分辨率水平上确定细胞内特定蛋白质的定位，对于理解这些蛋白质在诸如运动和细胞内运输等细胞过程中的功能至关重要。这种定位可以通过将透射电子显微镜（TEM）与免疫金标记相结合来实现。在这里，我们描述了一种包埋前间接金免疫标记方法，用于在盖玻片上生长的细胞中，使用不同大小金颗粒标记的二抗来定位两种不同的目标蛋白质。在这个方案中，细胞在被包埋于环氧树脂中进行超薄切片之前先进行免疫标记。该方案还包括优化超微结构与抗原保存之间平衡的策略、最小化非特异性抗体结合的步骤以及优化抗体穿透的步骤。
