Foster Timothy Paul
Department of Microbiology, Immunology & Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA, USA.
Methods Mol Biol. 2022;2422:85-124. doi: 10.1007/978-1-0716-1948-3_7.
The defining characteristic of eukaryotic cells is the segregation of critical cellular functions within various membrane bound cellular organelles, including the nucleus, endoplasmic reticulum, Golgi apparatus, lysosomes, and mitochondria. Cell biologists therefore have extensively utilized organelle specific counterstains to help identify the localization of specific proteins or other targets of interest in order to garner an understanding of either their potential functions or their effects on the cell. There currently is a wide array of fluorescent dyes and reagents that can be utilized in live and fixed cells to identify organelles, thereby creating challenges in both choosing between the plethora of options and optimizing their use. Here we present a discussion of commonly utilized commercially available organelle dyes and summarize the factors that influence selection of the various dyes for: a given organelle; live versus fixed cellular conditions; adaptation to a specific protocol; spectral multiplexing; or matching excitation/emission spectra to available imaging equipment. Also presented are recommended protocols for a typical example reagent that can be reliably utilized to visualize its target cellular organelle.
真核细胞的决定性特征是关键细胞功能在各种膜结合细胞器内的分隔，这些细胞器包括细胞核、内质网、高尔基体、溶酶体和线粒体。因此，细胞生物学家广泛利用细胞器特异性复染剂来帮助确定特定蛋白质或其他感兴趣靶点的定位，以便了解它们的潜在功能或它们对细胞的影响。目前有大量荧光染料和试剂可用于活细胞和固定细胞以识别细胞器，这在从众多选项中进行选择以及优化其使用方面都带来了挑战。在此，我们讨论常用的市售细胞器染料，并总结影响为以下情况选择各种染料的因素：特定细胞器；活细胞与固定细胞条件；适应特定实验方案；光谱复用；或将激发/发射光谱与可用成像设备相匹配。还介绍了一种典型示例试剂的推荐实验方案，该试剂可可靠地用于可视化其目标细胞器。
