Department of Biology, Reed College.
Department of Biology, Reed College; Committee on Development, Regeneration, and Stem Cell Biology, University of Chicago.
J Vis Exp. 2022 Feb 11(180). doi: 10.3791/63509.
Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.
斑马鱼具有显著的终身生长和再生能力。例如，在胚胎发生过程中建立的专门干细胞巢支持整个视觉系统的持续生长，包括眼睛和大脑。视网膜和视顶盖之间的协调生长确保了新神经元在眼睛和大脑中添加时，视网膜投射的精确映射。为了解决视网膜轴突是否为调节顶盖干细胞和祖细胞行为（如存活、增殖和/或分化）提供关键信息，有必要能够在同一动物和不同动物中比较神经支配和去神经支配的视顶盖叶。从活体幼鱼中切除一只眼睛，然后观察视顶盖，就可以实现这一目标。随附的视频演示了如何麻醉幼虫、电解锐化钨针，并使用它们来切除一只眼睛。接下来，它展示了如何从固定的斑马鱼幼虫中解剖大脑。最后，该视频概述了免疫组织化学的方案，并演示了如何将染色的胚胎安装在低熔点琼脂糖中进行显微镜检查。
