| Immunofluorescent Staining of Mouse Intestinal Stem Cells
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Author:
Date:
2016-02-20
[Abstract] Immunofluorescent staining of organoids can be performed to visualize molecular markers of cell behavior. For example, cell proliferation marked by incorporation of nucleotide (EdU), or to observe markers of intestinal differentiation including paneth cells, goblet cells, or enterocytes (see Figure 1). In this protocol we detail a method to fix, permeabilize, stain and mount intestinal organoids for analysis by immunofluorescent confocal microscopy.
Figure 1. A schematic depicting a crypt-villus forming organoid, and visualization of Paneth cells by immunofluorescence staining. Left: Small intestinal organoids grow as crypt-villus structures that contain all of the ...
[摘要] 可以进行类器官的免疫荧光染色以显现细胞行为的分子标志物。例如，通过掺入核苷酸（EdU）标记的细胞增殖，或观察肠分化的标志物，包括paneth细胞，杯状细胞或肠细胞（参见图1）。在这个协议中，我们详细的方法来修复，透化，染色和安装肠组织，通过免疫荧光共聚焦显微镜分析。
图1.描绘隐窝 - 绒毛形成类器官的示意图，通过免疫荧光染色观察Paneth细胞。肠器官类生长为含有所有肠的多种分化谱系的隐窝 - 绒毛结构。右：免疫荧光染色可用于显现器官类型中的单个细胞类型。通过染色溶菌酶（"Lyso，"Green）显示paneth细胞，其显示位于隐窝碱基的Paneth细胞。 F-肌动蛋白（红色）显示在上皮的顶端表面的隐窝结构，DAPI（蓝色）揭示细胞核。比例尺为25μm。
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| Immunoplaque Assay (Influenza Virus)
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Author:
Date:
2013-11-05
[Abstract] Despite developed long time ago, plaque assay is still the gold standard for viral titer quantification in modern virology. The standard crystal violet-based plaque assay relies on virus’ ability to induce cytopathic effect (CPE) which limits the assay to lytic viruses. Alternative viral quantification assays such as 50% tissue culture infectious assay (TCID50) and genetic material quantification by Q-PCR provide a different way of viral quantification with their own shortcoming. In here, we modified the fluorescent focus assay and developed an antibody-based immunoplaque assay which provides a reliable and reproducible viral quantification independent of CPE. Our assay not only allows accurate determination of viral titer, but also provides information on viral kinetics, ...
[摘要] 尽管发展很久以前，斑块测定仍然是现代病毒学病毒滴度量化的黄金标准。 标准的基于结晶紫的斑块测定依赖于病毒诱导细胞病变效应（CPE）的能力，其限制了对裂解病毒的测定。 替代性病毒定量测定如50％组织培养感染测定（TCID 50）和通过Q-PCR的遗传物质定量提供了具有其自身缺点的病毒定量的不同方式。 在这里，我们修改荧光焦点测定和开发基于抗体的免疫斑检测提供可靠和可重复的病毒定量独立于CPE。 我们的测定不仅允许病毒滴度的精确测定，而且提供关于病毒动力学，遗传稳定性和病毒种群的纯度的信息。
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