| Single Molecule RNA FISH in the Mammalian Oocyte
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Author:
Date:
2015-12-05
[Abstract] RNA fluorescence in situ hybridization is a method to localize and measure gene expression in individual cell or tissue. Using multiple specific fluorescently labeled oligonucleotides greatly increases signal-to-noise ratio and thus enables detection of single RNA molecule. Around forty different DNA oligonucleotides designed to common RNA target and labeled with single fluorophore at 3´ terminus hybridizes with target RNA in fixed cells. We adapt this method to visualize target RNA in the mammalian oocyte. The ability to detect single transcript in the mammalian oocyte was challenging due to its large cell size. This method consists of four simple steps: fixation, permeabilization, hybridization and imaging. The protocol is adapted to this large nonattached cell to visualize ...
[摘要] RNA荧光原位杂交是定位和测量个体细胞或组织中的基因表达的方法。 使用多个特异性荧光标记的寡核苷酸大大增加信噪比，从而使得能够检测单个RNA分子。 大约四十个不同的DNA寡核苷酸设计为常见的RNA靶标，并在3'端用单个荧光团标记，与固定细胞中的靶RNA杂交。 我们适应这种方法可视化目标RNA在哺乳动物卵母细胞。 在哺乳动物卵母细胞中检测单个转录物的能力由于其大的细胞大小而具有挑战性。 该方法由四个简单的步骤组成：固定，预稳定化，杂交和成像。 该方案适应这种大的非附着细胞以显现母体RNA。
各种荧光团的组合允许检测更多的RNA靶标。 该方法可以与细胞器标记一起使用或用免疫荧光方案扩增。
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| ELISA on Virus-Infected Cells
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Author:
Date:
2014-05-20
[Abstract] The gammaherpesvirus murid herpesvirus 4 (MuHV-4) enters cells by endocytosis from the cell surface and fusion of the viral envelope with the membrane of late endosomes. The viral envelope glycoproteins undergo antigenic changes both upon virion endocytosis and upon fusion of the viral envelope with the endosomal membrane. These changes in virion antigenicity during virus entry were first described by immunofluorescence of infected cells. Although immunofluorescence provides valuable information on the subcellular distribution of the viral glycoproteins, the quantification of immunofluorescence signals in a large number of cells is not only dependent on relatively expensive microscopy equipment, but is also relatively time-consuming. In order to quantify the antigenicity of MuHV-4 virions ...
[摘要] γ疱疹病毒鼠疱疹病毒4（MuHV-4）通过从细胞表面的内吞作用进入细胞并且病毒包膜与晚期内体膜的融合。病毒包膜糖蛋白在病毒颗粒内吞后和病毒包膜与内体膜融合时经历抗原性改变。病毒进入期间病毒体抗原性的这些变化首先通过感染细胞的免疫荧光来描述。尽管免疫荧光提供了关于病毒糖蛋白的亚细胞分布的有价值的信息，但是大量细胞中免疫荧光信号的定量不仅依赖于相对昂贵的显微镜设备，而且相对耗时。为了以可靠，以及时间和成本有效的方式定量进入NMuMG上皮细胞的MuHV-4病毒粒子的抗原性，我们开发了具有感染细胞作为固相的ELISA。在该测定中，细胞在96孔组织培养板上生长，在4℃暴露于病毒粒子，然后在37℃孵育，允许病毒粒子内吞。细胞在病毒体结合后直接在4℃固定，或在37℃孵育后固定。在随后的透化作用之后，将细胞与对病毒包膜糖蛋白特异性的单克隆抗体温育，然后用碱性磷酸酶偶联的二抗检测。在用磷酸硝基苯酯底物孵育细胞时，在常规ELISA酶标仪上测量吸光度。讨论了不同的数据解释方式。
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