Localization of Hippo Signaling Components in Drosophila by Fluorescence and Immunofluorescence.
Localization of Hippo Signaling Components in Drosophila by Fluorescence and Immunofluorescence.
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DOI：
10.1007/978-1-4939-8910-2_5
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发表时间：
2018-12
影响因子：
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通讯作者：
C. Rauskolb;Kenneth D. Irvine
中科院分区：
文献类型：
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作者：
C. Rauskolb;Kenneth D. Irvine
体内蛋白质水平和定位的可视化对于分析和阐明河马信号机制及其在不同组织中的作用至关重要。最好的方法是使用荧光标记对蛋白质进行成像。蛋白质的荧光标记可以通过直接连接到固有的荧光蛋白，如GFP，或通过使用连接到荧光染料的抗体来实现。果蝇的免疫荧光成像通常是从解剖和固定样本组织开始，然后是针对感兴趣的蛋白质的一次抗体和针对一次抗体的一系列洗涤和孵育，以及针对一次抗体的染料标记的二次抗体。紧随其后的可能是标记细胞成分的荧光染料，例如标记细胞核的DNA标记染料。染色和洗涤完成后，将样品放置在安装介质中，转移到显微镜载玻片上，并在共焦显微镜上成像。
Visualization of in vivo protein levels and localization is essential to analysis and elucidation of Hippo signaling mechanisms and its roles in diverse tissues. This is best done by imaging proteins using fluorescent labels. Fluorescent labeling of a protein can be achieved by direct conjugation to an intrinsically fluorescent protein, like GFP, or by use of antibodies conjugated to fluorescent dyes. Immunofluorescence imaging inDrosophilatypically begins with dissection and fixation of a sample tissue, followed by a series of washes and incubations with primary antibodies, directed against proteins of interest, and dye-labeled secondary antibodies, directed against the primary antibodies. This may be followed by fluorescent dyes that label cellular components, such as DNA-labeling dyes to mark nuclei. After staining and washing is completed, samples are placed in a mounting media, transferred to a microscope slide, and imaged on a confocal microscope.