Ashley James, Carrillo Robert A
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA.
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA
Cold Spring Harb Protoc. 2025 Jul 1;2025(7):pdb.prot108499. doi: 10.1101/pdb.prot108499.
One of the challenges of studying synaptic structure and function is accessibility. Some of the earliest readily identifiable and accessible synapses were from the frog and various arthropods. To address questions regarding mechanisms that underlie synaptic development and function, genetically tractable systems were required, and researchers turned to the embryonic/larval neuromuscular preparation. embryos are transparent and can be labeled with antibodies or probes and imaged in whole-mount preparation for structural analysis. Embryos can also be dissected to visualize the entire body wall musculature as well as finer details including live protein trafficking and protein-protein interactions. Whereas younger dissected embryos can be mounted directly onto charged slides, more mature embryos and larvae develop a cuticle that impedes this adherence, so different techniques must be applied. In this protocol, we detail how to manufacture dissection tools and collect embryos, and discuss the individual steps of dissecting late-stage embryos, early first-instar larvae, and late-stage third-instar larvae.
研究突触结构与功能面临的挑战之一是可及性。一些最早易于识别且可触及的突触来自青蛙和各种节肢动物。为了解决有关突触发育和功能潜在机制的问题，需要遗传上易于操作的系统，研究人员于是转向胚胎/幼虫神经肌肉标本。胚胎是透明的，可以用抗体或探针标记，并在整装标本中成像以进行结构分析。胚胎也可以解剖以观察整个体壁肌肉组织以及更精细的细节，包括活蛋白运输和蛋白质-蛋白质相互作用。较年幼的解剖胚胎可以直接安装在带电荷的载玻片上，而更成熟的胚胎和幼虫会形成一层角质层，阻碍这种附着，因此必须应用不同的技术。在本方案中，我们详细介绍如何制作解剖工具和收集胚胎，并讨论解剖晚期胚胎、一龄幼虫早期和三龄幼虫晚期的各个步骤。
