Rehm E Jay, Hannibal Roberta L, Chaw R Crystal, Vargas-Vila Mario A, Patel Nipam H
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3140, USA.
Cold Spring Harb Protoc. 2009 Jan;2009(1):pdb.prot5129. doi: 10.1101/pdb.prot5129.
The great diversity of arthropod body plans, together with our detailed understanding of fruit fly development, makes arthropods a premier taxon for examining the evolutionary diversification of developmental patterns and hence the diversity of extant life. Crustaceans, in particular, show a remarkable range of morphologies and provide a useful outgroup to the insects. The amphipod crustacean Parhyale hawaiensis is becoming established as a model organism for developmental studies within the arthropods. This protocol provides a simplified protocol for antibody staining of P. hawaiensis embryos. The method also works well for other arthropods and phyla. Fixed embryos are rehydrated, washed, blocked with normal goat serum, and incubated overnight with primary antibody. Embryos are then washed and incubated with a peroxidase-conjugated secondary antibody that binds to the primary antibody. A subsequent histochemical reaction produces a black stain in those cells where antibodies have localized.
节肢动物身体结构的巨大多样性，加上我们对果蝇发育的详细了解，使节肢动物成为研究发育模式进化多样化以及现存生命多样性的首要类群。特别是甲壳类动物，展现出了显著的形态范围，并为昆虫提供了一个有用的外类群。夏威夷片脚类甲壳动物Parhyale hawaiensis正在成为节肢动物发育研究的模式生物。本方案提供了一种简化的夏威夷片脚类胚胎抗体染色方案。该方法对其他节肢动物和门也同样适用。固定后的胚胎进行复水、洗涤，用正常山羊血清封闭，然后与一抗孵育过夜。接着胚胎被洗涤，并与结合一抗的过氧化物酶偶联二抗孵育。随后的组织化学反应会在抗体定位的细胞中产生黑色染色。
