Dongre Shivali, Hilbert Lennart, Vastenhouw Nadine L
Center for Integrative Genomics, University of Lausanne, Quartier Sorge, Lausanne, Switzerland.
Department of Systems Biology/Bioinformatics, Zoological Institute, Karlsruhe Institute of Technology, Karlsruhe, Germany.
Methods Mol Biol. 2025;2923:89-117. doi: 10.1007/978-1-0716-4522-2_7.
The transcriptional machinery of a cell is often not homogenously distributed but rather forms clusters in the nucleus. These clusters are important for gene expression, but how they form and function is often not clear. The zebrafish embryo provides an excellent system to study these clusters of transcriptional machinery, because embryos are transparent and develop rapidly, making it easy to track proteins as they cluster and perform their function. Here, we provide a protocol for how to image nuclear clusters in living zebrafish embryos. The protocol includes information on the selection and encoding of proteins and fluorophores, embryo-embedding for live-cell microscopy, the use of a spinning disk microscope, staging of embryos post image acquisition, and image analysis. While the protocol is written in the context of our work with early zebrafish embryos, many of the tools will be useful in other contexts.
细胞的转录机制通常并非均匀分布，而是在细胞核中形成簇。这些簇对基因表达很重要，但其形成和功能往往尚不清楚。斑马鱼胚胎提供了一个研究这些转录机制簇的绝佳系统，因为胚胎是透明的且发育迅速，便于追踪蛋白质在聚集并发挥功能时的情况。在此，我们提供了一个在活体斑马鱼胚胎中对细胞核簇进行成像的方案。该方案包括有关蛋白质和荧光团的选择与编码、用于活细胞显微镜观察的胚胎包埋、转盘显微镜的使用、图像采集后胚胎的分期以及图像分析等信息。虽然该方案是基于我们对早期斑马鱼胚胎的研究工作编写的，但其中许多工具在其他情况下也会有用。
