Hammond-Weinberger Dena R, ZeRuth Gary T
Department of Biological Sciences, Murray State University.
Department of Biological Sciences, Murray State University;
J Vis Exp. 2020 Jan 29(155). doi: 10.3791/60575.
Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.
免疫组织化学是一种广泛应用的技术，用于探究正常发育和疾病状态下蛋白质的表达及定位。尽管许多免疫组织化学方案已针对哺乳动物组织和组织切片进行了优化，但这些方案通常需要针对非哺乳动物模式生物进行修改和优化。斑马鱼越来越多地被用作基础、生物医学和转化研究中的模型系统，以研究发育过程的分子、遗传和细胞生物学机制。斑马鱼作为模型系统具有许多优势，但也需要改进技术以实现最佳的蛋白质检测。在此，我们提供了斑马鱼胚胎和幼体全胚胎荧光免疫组织化学的方案。该方案还描述了几种可采用的不同封片策略，并概述了每种策略的优缺点。我们还描述了对该方案的修改，以实现全胚胎组织中显色底物的检测以及幼体组织切片中的荧光检测。该方案广泛适用于许多发育阶段和胚胎结构的研究。
