| Staining of Membrane Receptors with Fluorescently-labeled DNA Aptamers for Super-resolution Imaging
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Author:
Date:
2017-09-05
[Abstract] One of the most prominent applications of fluorescent super-resolution microscopy is the study of nanodomain arrangements of receptors and the endocytic pathway. Staining methods are becoming crucial for answering questions on the nanoscale, therefore, the use of small and monovalent affinity probes is of great interest in super-resolution microscopy with biological samples. One kind of affinity probe is the aptamer. Aptamers are single DNA or RNA sequences that bind with high affinity to their targets and due to their small size they are able to (i) place the fluorophore in close proximity to the protein of interest and (ii) bind to most of the protein of interest overcoming the steric hindrance effect, resulting in better staining density. Here we describe a detailed protocol with which ...
[摘要] 荧光超分辨率显微镜的最突出的应用之一是研究纳米结构的受体和内吞途径。染色方法对于回答纳米尺度的问题变得至关重要，因此，使用小型和单价亲和探针在具有生物样品的超分辨显微镜中是非常有意义的。一种亲和力探针是适体。适配体是以高亲和力结合其靶标的单个DNA或RNA序列，并且由于它们的小尺寸，它们能够（i）将荧光团置于感兴趣的蛋白质附近，并且（ii）与大部分蛋白质结合有利于克服空间位阻效应，导致更好的染色密度。在这里，我们描述一个详细的协议，使用适配体染色活细胞，并用刺激的排放消耗（STED）显微镜对其进行成像。在该方案中，染色用市售适用于靶向表皮生长因子受体（EGFR），人表皮生长因子受体2（HER2或ErbB2）和ephrin-A受体2（Epha2）的适体进行。由于适配体可与大部分受欢迎的荧光团偶联，因此我们认为本文介绍的方法可扩展到目前超分辨率显微镜技术的绝大多数。
【背景】超分辨率成像技术的最新进展已经导致搜索更精确的方法来标记细胞元件。衍射无限成像仪器提供优异的分辨率，然而标准样品染色方法，如免疫染色，缺乏检测细胞元素所必需的精度。由于它们的大尺寸（长度约15nm）和高分子量（〜150kDa），抗体可以很差地渗透到生物样品中。另外，一次/二次抗体复合物将荧光团放置在远离靶的大约25nm处，从而损害检测精度。此外，由于初级/二级抗体复合物的大尺寸，由于空间位阻（Fornasiero和Opazo，2015），较小部分的靶标可以被标记。这导致较低的标记密度，这是超分辨率显微镜的关键参数，特别是在识别和描述纳米结构时。为了克服这些问题，近年来已经测试了与单个靶（单价）结合的小的亲和力探针，如适体，亲和体或纳米体系（Rothbauer ...
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| Isolation and Culture of the Islets of Langerhans from Mouse Pancreas
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Author:
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2016-06-20
[Abstract] The islets of Langerhans are clusters of endocrine cells located within the pancreas. Insulin-producing beta cells are the major cell type within islets, with glucagon-producing alpha cells and somatostatin-producing delta cells the other major cell types. The beta cells are the target of immune-mediated destruction in type 1 diabetes (Graham et al., 2012). Failure of beta cell function accompanied by loss of beta cell mass is also a feature of type 2 diabetes (Wali et al., 2013). Therefore studying the biology of pancreatic islets is important to understand the pathogenesis of diabetes and to develop new therapies. Here we describe the isolation of mouse islets. This requires gentle enzymatic and mechanical digestion of the exocrine tissue and density gradient ...
[摘要] 朗格汉斯岛是位于胰腺内的内分泌细胞簇。胰岛素生成β细胞是胰岛内的主要细胞类型，产生胰高血糖素的α细胞和生长抑素生成的δ细胞是其他主要细胞类型。 β细胞是免疫介导的1型糖尿病破坏的靶标（Graham等，2012）。 β细胞功能衰竭伴有β细胞量的丧失也是2型糖尿病的特征（Wali等，2013）。因此，研究胰岛的生物学对于了解糖尿病的发病机制和开发新的疗法是重要的。这里我们描述了小鼠胰岛的分离。这需要温和的酶促和机械消化外分泌组织和密度梯度分离（Chong et al。，2004; Liu和Shapiro，1995; Thomas et al。，1998）。然后，我们将描述胰岛如何可以整体培养或分散到单个细胞中，用于各种体外和体内分析。使用该协议可靠地导致200-400个胰岛的隔离，这取决于鼠标的应变。
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| RNA Isolation and Northern Blot Analysis
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Author:
Date:
2014-03-20
[Abstract] The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Here, we examine ATF3, ATF4, and GADD153 gene expression profiles by northern blot in Vero cells and H1299 cells after IBV infection. RNA was extracted in IBV (infectious bronchitis virus) infected cells and electrophoresis was used to separate the RNA sample. RNA was transferred from the electrophoresis gel to the blotting membrane by capillary transfer. Specific mRNA was detected with hybridization probes complementary to part of target sequence. The probes were prepared by RT-PCR and labeled by ...
[摘要] Northern印迹是在分子生物学研究中用于通过检测样品中的RNA来研究基因表达的技术。 使用Northern印迹，可以在分化，形态发生以及异常或疾病状况期间观察到特定的基因表达水平。 在这里，我们检查ATF3，ATF4和GADD153基因表达谱通过北部污点在Vero细胞和H1299细胞后IBV感染。 在IBV（感染性支气管炎病毒）感染的细胞中提取RNA，并使用电泳分离RNA样品。 通过毛细管转移将RNA从电泳凝胶转移到印迹膜上。 使用与靶序列的一部分互补的杂交探针检测特异性mRNA。 通过RT-PCR制备探针，并使用DIG标记试剂盒通过地高辛（DIG）标记。
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