Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos.
Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos.
复制标题
整个斑马鱼胚胎纤毛器官中的磷酸化表位的免疫染色。
DOI：
10.3791/53747
复制
发表时间：
2016
期刊：
影响因子：
--
通讯作者：
R. Tombes
中科院分区：
文献类型：
--
作者：
Sarah C. Rothschild;Ludmila Francescatto;R. Tombes
细胞的快速增殖、基因的组织特异性表达和信号网络的出现是所有脊椎动物早期胚胎发育的特征。发育胚胎中信号的动力学和位置（即使是在单细胞内）补充了重要发育基因的识别。免疫染色技术已被证明可以定义小至初级纤毛的结构中的细胞内和整个动物信号的动力学。使用激光扫描共焦复合显微镜进行图像固定、成像和处理的技术可在短短 36 小时内完成。斑马鱼（Danio rerio）对于寻求在脊椎动物物种中进行研究的研究人员来说是一种理想的生物，这种脊椎动物物种负担得起且与人类疾病相关。基因敲除或基因敲低必须通过实际蛋白质产物的损失来确认。可以使用此处描述的技术来实现蛋白质损失的这种确认。信号传导途径的线索也可以通过使用与经过磷酸化翻译后修饰的蛋白质发生反应的抗体来破译。因此，保留和优化表位的磷酸化状态对于该测定至关重要，并通过该方案完成。这项研究描述了在发育的前 72 小时内固定胚胎并将各种相关表位与库普弗囊泡 (KV)、肾脏和内耳中的纤毛共定位的技术。这些技术很简单，不需要解剖，并且可以在相对较短的时间内完成。将共焦图像堆栈投影到单个图像中是呈现这些数据的有用方法。
The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr. Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol. This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer's Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.
影响因子：
13.6
作者：
Obara, Tomoko;Mangos, Steven;Drummond, Iain A.
通讯作者：
Drummond, Iain A.
CaMK-II 激活对于斑马鱼内耳发育至关重要，并通过 Delta-Notch 信号传导发挥作用。
影响因子：
2.7
作者：
Rothschild,SarahC;Lahvic,Jamie;Francescatto,Ludmila;McLeod,JamieJA;Burgess,ShawnM;Tombes,RobertM
通讯作者：
Tombes,RobertM