Ctenophore whole-mount antibody staining.
Ctenophore whole-mount antibody staining.
复制标题
栉水母整体抗体染色。
DOI：
10.1101/pdb.prot5086
复制
发表时间：
2008
期刊：
影响因子：
--
通讯作者：
M. Martindale
中科院分区：
文献类型：
--
作者：
K. Pang;M. Martindale
栉水母，或梳水母，是一组海洋动物，其独特的生物学特征和系统发育的位置，使他们了解动物进化的关键类群。几乎所有的栉水母都有一些特征，包括双辐射对称，由连接的纤毛组成的梳行，顶端感觉器官和两个带有专门的粘附细胞的触手。迄今为止研究的所有栉水母都有相同的定型卵裂程序，并经历一个称为cydippid幼虫的特定发育阶段，在此之后，成虫结构发育并在物种之间差异很大;这对于比较研究特别有用。由于易于收集胚胎，它们的大小（在某些物种中可达1毫米）和它们的快速发育，栉水母一直是实验胚胎学家的吸引力动物。该方案描述了如何固定栉水母胚胎及其cydippid幼虫用于抗体染色。一旦样品被固定，将组织与针对感兴趣的表位的抗体一起孵育。然后，与荧光分子缀合的第二抗体揭示表位的表达模式。荧光显微镜用于可视化和记录信号。该方案还包括用鬼笔环肽的荧光衍生物染色或复染的方法，其揭示肌肉和细胞边界中的F-肌动蛋白。虽然该方案侧重于胚胎和幼虫样本，但该技术也可应用于成体组织。
INTRODUCTIONCtenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. Because of the ease of embryo collection, their size (up to 1 mm in some species), and their rapid development, ctenophores have been attractive animals for experimental embryologists. This protocol describes how to fix ctenophore embryos and their cydippid larvae for antibody staining. Once the samples have been fixed, tissues are incubated with an antibody to the epitope of interest. A secondary antibody conjugated to a fluorescent molecule then reveals the expression pattern of the epitope. Fluorescent microscopy is used to visualize and document the signal. The protocol also includes methods for staining or counterstaining with a fluorescent derivative of phalloidin, which reveals F-actin in muscles and cell borders. Although the protocol focuses on embryonic and larval samples, the technique can also be applied to adult tissues.