Ethell Douglas W, Cameron D Joshua
Molecular Neurobiology, Western University of Health Sciences; Graduate College of Biomedical Sciences, Western University of Health Sciences; College of Osteopathic Medicine of the Pacific, Western University of Health Sciences;
Molecular Neurobiology, Western University of Health Sciences; College of Optometry, Western University of Health Sciences.
J Vis Exp. 2014 Apr 22(86):50417. doi: 10.3791/50417.
Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.
斑马鱼是用于体内研究发育生物学和病理学的强大工具。斑马鱼胚胎体积小且相对透明，这使得它们在诸如心脏和血管发育等过程的视觉检查中特别有用。在最近的几项研究中，利用共聚焦显微镜，使用在血管内皮细胞中表达增强绿色荧光蛋白（EGFP）的转基因斑马鱼对大脑和视网膜中的复杂血管网络进行成像和分析。本文提供了制备、处理和对表达增强绿色荧光蛋白（EGFP）的斑马鱼胚胎进行成像的方法，然后生成脑血管系统的全面三维渲染图。实验方案包括胚胎处理、共聚焦成像以及保留EGFP荧光的固定方案。此外，还提供了获取脑血管结构高质量图像的有用提示，例如在不损伤附近神经组织的情况下摘除眼睛。讨论了共聚焦成像的潜在陷阱，以及使用免费开源软件从共聚焦图像堆栈生成三维重建所需的步骤。
