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Dulbecco’s Phosphate Buffered Saline

Dulbecco磷酸盐缓冲盐水

Company: Sigma-Aldrich
Catalog#: D8662
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Immunogold Electron Microscopy of the Autophagosome Marker LC3
Author:
Date:
2017-12-20
[Abstract]  Even though autophagy was firstly observed by transmission electron microscopy already in the 1950s (reviewed in Eskelinen et al., 2011), nowadays this technique remains one of the most powerful systems to monitor autophagic processes. The autophagosome, an LC3-positive double membrane structures enclosing cellular materials, represents the key organelle in autophagy and its simple visualization and/or numeration allow to draw important conclusions about the autophagic flux. Therefore, the accurate identification of autophagosomes is crucial for a comprehensive and detailed dissection of autophagy. Here we present a simple protocol to identify autophagosomes by transmission electron microscopy coupled to immunogold labeling of LC3 starting from a relatively low cell number, which ... [摘要]  尽管早在20世纪50年代就已经通过透射电子显微镜观察了自噬(在Eskelinen等人2011年的综述中),但是现在这种技术仍然是监测自噬过程的最强大的系统之一。 自噬体是包含细胞物质的LC3阳性双层膜结构,代表了自噬的关键细胞器,其简单的可视化和/或计数允许得出关于自噬流的重要结论。 因此,准确鉴定自噬体对自噬的全面和详细的分析至关重要。 在这里我们提出一个简单的协议,以确定autophagosomes透射电子显微镜耦合LC3的免疫金标记从一个相对较低的细胞数量,我们最近开发遵循病毒介导的人类癌变期间的自噬途径。

【背景】自噬体代表了macroautophagy的关键结构,这是一种细胞胞质成分的分解代谢系统。巨自噬(或简单地自吞噬)由吞噬细胞的形成引发,所述吞噬细胞能够自身扩张吞噬细胞器和蛋白质,所述蛋白质最终闭合在螯合成分周围形成被称为自噬体的细胞器。接下来,在成熟过程中,自噬体可以与溶酶体融合以形成自溶酶体,其中被捕获的物质被位于溶酶体限制性膜中的泵降解并再循环回(Glick et al。,2010)。

由于自噬功能障碍与各种人类疾病,病毒感染,神经退行性疾病,免疫功能和癌症有关(Schneider and ...

Staining of Membrane Receptors with Fluorescently-labeled DNA Aptamers for Super-resolution Imaging
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 ...

Analysis of the Virulence of Uropathogenic Escherichia coli Strain CFT073 in the Murine Urinary Tract
Author:
Date:
2017-02-05
[Abstract]  This urinary tract infection model was used to monitor the efficacy of a new virulence factor of the uropathogenic Escherichia coli strain CFT073 in vivo. The new virulence factor which we designated TIR-containing protein C (TcpC) blocks Toll-like receptor signaling and the NLRP3 inflammasome signaling cascade by interacting with key components of both pattern recognition receptor systems (Cirl et al., 2008; Waldhuber et al., 2016). We infected wild type and knock-out mice with wildtype CFT073 and a mutant CFT073 strain lacking tcpC. This protocol describes how the mice were infected, how CFT073 was prepared and how the infection was monitored. The protocol was derived from our previously published work and allowed us to demonstrate that TcpC ... [摘要]  该尿路感染模型被用于监测新生的致病性大肠杆菌菌株CFT073在体内的功效。我们指定含TIR的蛋白C(TcpC)的新的毒力因子通过与模式识别受体系统的关键组分相互作用来阻断Toll样受体信号传导和NLRP3炎性信号级联反应(Cirl等人)。 ,2008; Waldhuber等人,2016)。我们用野生型CFT073和缺乏tcpC的突变体CFT073菌株感染野生型和敲除小鼠。该协议描述了小鼠如何感染,如何制备CFT073以及如何监测感染。该方案源于我们以前发表的工作,并允许我们证明TcpC是一种强大的毒力因子,通过增加CFT073在尿液和肾脏中的细菌负担。此外,TcpC负责肾脓肿的发展,因为感染具有野生型但不是tcpC的缺乏CFT073突变体的小鼠引起这种并发症。

背景 尿路感染(UTIs)是全世界最常见的细菌感染(Dielubanza和Schaeffer,2011),主要是由欧洲病原大肠杆菌(UPEC)引起的(Zhang和Foxman,2003)。复发性感染率高(Dielubanza和Schaeffer,2011),抗生素抗性E的出现也有所增加。大肠杆菌菌株(Eurosurveillance editorial,2015)。因此,为了开发新的治疗剂,对宿主和细菌因子对尿路感染病理生理学的了解具有很高的相关性。
 鼠类UTI模型系统是主要使用的动物模型系统,用于研究UPEC分离株和细菌 ...

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