Parton Richard M, Vallés Ana Maria, Dobbie Ian M, Davis Ilan
Cold Spring Harb Protoc. 2010 Apr;2010(4):pdb.prot5403. doi: 10.1101/pdb.prot5403.
The fruit fly Drosophila melanogaster is an important model for basic research into the molecular mechanisms underlying cell function and development, as well as a major biomedical research tool. A significant advantage of Drosophila is the ability to apply live cell imaging to a variety of living tissues that can be dissected and imaged in vivo, ex vivo, or in vitro. For example, such imaging can be used for visual genetic screens such as analysis of morphological characteristics or of the distribution of fluorescently tagged proteins in living embryos. Indeed, Drosophila embryos have proven to be a useful model system for studying a variety of cellular processes such as asymmetric division, migration, wound healing, apoptosis, and fasciculation, as well as for tracking lipid droplet motility, protein recycling, fast mRNA transport, and the movement of chromosomal loci within individual cells. A crucial first step before imaging is preparation of the experimental material to ensure physiological relevance and to achieve the best conditions for image quality. Because it contributes to autofluorescence and lack of transparency, it is useful to remove the chorion before imaging by treating the embryos with bleach. This protocol describes the collection and mounting of Drosophila embryos for live cell imaging.
果蝇黑腹果蝇是研究细胞功能和发育背后分子机制的基础研究的重要模型,也是一种主要的生物医学研究工具。果蝇的一个显著优势是能够将活细胞成像应用于各种可以在体内、体外或离体状态下进行解剖和成像的活组织。例如,这种成像可用于视觉遗传筛选,如分析活胚胎中的形态特征或荧光标记蛋白的分布。事实上,果蝇胚胎已被证明是研究多种细胞过程的有用模型系统,如不对称分裂、迁移、伤口愈合、细胞凋亡和束状化,以及追踪脂滴运动、蛋白质循环利用、快速mRNA运输和单个细胞内染色体位点的移动。成像前至关重要的第一步是制备实验材料,以确保生理相关性并实现最佳图像质量条件。由于它会导致自发荧光和透明度不足,在成像前用漂白剂处理胚胎以去除卵壳是很有用的。本方案描述了用于活细胞成像的果蝇胚胎的收集和固定。