| 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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| Dictyostelium Cultivation, Transfection, Microscopy and Fractionation
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Author:
Date:
2015-06-05
[Abstract] The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. In addition the fractionation of cells and isolation of organelles or known compartments can often verify any subcellular localisation and the use of tagged proteins as bait for the immunoprecipitation of material from cell fractions can identify specific binding partners and multiprotein complexes thereby helping assign a function to the tagged protein. We have successfully applied these techniques to the Dictyostelium discoideum protein TSPOON that is part of an ancient ...
[摘要] 使用更复杂的显微镜，活细胞中荧光标记的蛋白质的实时可视化大大增加了我们对基本生理过程如细胞运动，趋化性，细胞分裂和膜运输过程中关键蛋白质动力学的了解。此外，细胞的分级和分离细胞器或已知的隔室通常可以验证任何亚细胞定位，并且使用标记的蛋白质作为诱饵用于来自细胞部分的物质的免疫沉淀可以鉴定特异性结合配偶体和多蛋白复合物，从而有助于赋予功能标记蛋白。我们已经成功地将这些技术应用于作为古代异构六聚体膜转运复合物的一部分的盘基网柄菌discoideum蛋白TSPOON（Hirst等，2013）。 ...
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| Hypoxia Studies with Pimonidazole in vivo
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Author:
Date:
2014-10-05
[Abstract] Therapy-induced hypoxia drives changes in the tumor microenvironment that contribute to the poor response to therapy. Hypoxia is capable of driving the expression and/or activation of specific signaling cascades (e.g., c-Met, Axl, CTGF), the recruitment of tumor promoting immune cells, and the induction of cell survival pathways including autophagy (Phan et al., 2013; Hu et al., 2012; Ye et al., 2010). We have recently shown that anti-VEGF therapy-induced hypoxia can result in changes in the extracellular matrix that contribute to the aggressiveness of tumors post therapy (Aguilera et al., 2014). Importantly, therapies that induce hypoxia do not always increase epithelial plasticity and tumor aggressiveness (Ostapoff et al., 2013; ...
[摘要] 治疗诱导的缺氧驱动肿瘤微环境中的变化，其导致对治疗的差的反应。缺氧能够驱动特异性信号级联（例如，c-Met，Axl，CTGF）的表达和/或活化，肿瘤促进免疫细胞的募集和细胞存活途径的诱导，包括自噬（Phan等人，2013; Hu等人，2012; Ye等人，2010）。我们最近已经显示，抗VEGF治疗诱导的缺氧可以导致细胞外基质的变化，其有助于治疗后肿瘤的侵袭性（Aguilera等人，2014）。重要的是，诱导缺氧的治疗不总是增加上皮可塑性和肿瘤侵袭性（Ostapoff等人，2013; Cenik等人，2013）。我们已经使用哌莫硝唑来评价肿瘤中的缺氧，并且在本文中为这种有用的工具提供详细的方案来询问体内缺氧水平。
Hypoxyprobe TM （哌莫硝唑盐酸盐）免疫组织化学分析方法的实用性允许评估不同组织以及细胞类型中的缺氧。哌莫硝唑是2-硝基咪唑，其在缺氧细胞中特异性地被还原活化，并与蛋白质，肽和氨基酸中的巯基形成稳定的加合物（Cenik等人，2013; Arnold等人。，2010; Raleigh和Koch，1990; Raleigh等人，1998）。此外，检测到的吡莫硝唑的量与肿瘤内的缺氧水平成正比
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