Plant Development and Electron Microscopy, Department of Biology I, Munchen, Germany.
LOEWE Centre for Synthetic Microbiology (SYNMIKRO), Philipps-Universität Marburg, Marburg, Germany.
Microsc Res Tech. 2020 Jun;83(6):691-705. doi: 10.1002/jemt.23459. Epub 2020 Feb 14.
For nearly 50 years immunogold labeling on ultrathin sections has been successfully used for protein localization in laboratories worldwide. In theory and in practice, this method has undergone continual improvement over time. In this study, we carefully analyzed circulating protocols for postembedding labeling to find out if they are still valid under modern laboratory conditions, and in addition, we tested unconventional protocols. For this, we investigated immunolabeling of Epon-embedded cells, immunolabeling of cells treated with osmium, and the binding behavior of differently sized gold particles. Here we show that (in contrast to widespread belief) immunolabeling of Epon-embedded cells and of cells treated with osmium tetroxide is actually working. Furthermore, we established a "speed protocol" for immunolabeling by reducing antibody incubation times. Finally, we present our results on three-dimensional immunogold labeling.
近 50 年来，免疫金标记技术在超薄片上的应用已成功地在全球范围内的实验室中用于蛋白质定位。从理论和实践上看，这种方法随着时间的推移不断得到改进。在这项研究中，我们仔细分析了包埋后标记的循环方案，以了解它们在现代实验室条件下是否仍然有效，此外，我们还测试了非常规方案。为此，我们研究了包埋在环氧树脂中的细胞的免疫标记、用锇处理的细胞的免疫标记以及不同大小的金颗粒的结合行为。在这里，我们表明（与普遍的看法相反），对环氧树脂包埋的细胞和用四氧化锇处理的细胞进行免疫标记实际上是可行的。此外，我们通过减少抗体孵育时间建立了一种免疫标记的“快速方案”。最后，我们展示了三维免疫金标记的结果。
