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Uranyl Acetate powder

乙酸铀二水合物

Company: Electron Microscopy Sciences
Catalog#: 22400
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FM1-43 Photoconversion and Electron Microscopy Analysis at the Drosophila Neuromuscular Junction
Author:
Date:
2017-09-05
[Abstract]  We developed a protocol for photoconversion of endocytic marker FM1-43 followed by electron microscopy analysis of synaptic boutons at the Drosophila neuromuscular junction. This protocol allows detection of stained synaptic vesicle even when release rates are very low, such as during the spontaneous release mode. The preparations are loaded with the FM1-43 dye, pre-fixed, treated and illuminated to photoconvert the dye, and then processed for conventional electron microscopy. This procedure enables clear identification of stained synaptic vesicles at electron micrographs. [摘要]  我们开发了内吞标记FM1-43的光转换方案,然后在果蝇神经肌肉接头处进行突触引物的电子显微镜分析。 即使在释放速率非常低时,例如在自发释放模式期间,该方案允许检测染色的突触小泡。 该制剂装载有FM1-43染料,经预先固定,处理和照射,以使染料转变为染料,然后进行常规电子显微镜处理。 该方法能够在电子显微照片下清楚鉴定染色的突触小泡。
【背景】神经元发射体通过突触小泡与神经元质膜的融合而释放。囊泡可以自发融合或响应动作电位。随后,囊泡通过内吞作用获得回收。通过分子生物学,电生理学和显微镜的工具广泛研究了突触小泡回收的分子机制(Slepnev和De Camilli,2000; Sudhof,2004; Rizzoli和Betz,2005; Kavalali,2006)。加载内参标记FM1-43与染料光转换耦合,然后进行电子显微镜分析是一种强大的技术,允许调查和测量回收囊泡池(Harata et al。,2001; Schikorski and Stevens,2001; Rizzoli和Betz, 2004)。果蝇神经肌肉接头(NMJ)是具有明确定义的突触引物的有利制剂,其能够快速产生具有突变突触蛋白的细胞系和严格评估囊泡回收池(Akbergenova和Bykhovskaia,2009; ...

Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System
Author:
Date:
2017-06-20
[Abstract]  Efficient delivery of oligonucleotide therapeutics, i.e., siRNAs, to the central nervous system represents a significant barrier to their clinical advancement for the treatment of neurological disorders. Small, endogenous extracellular vesicles were shown to be able to transport lipids, proteins and RNA between cells, including neurons. This natural trafficking ability gives extracellular vesicles the potential to be used as delivery vehicles for oligonucleotides, i.e., siRNAs. However, robust and scalable methods for loading of extracellular vesicles with oligonucleotide cargo are lacking. We describe a detailed protocol for the loading of hydrophobically modified siRNAs into extracellular vesicles upon simple co-incubation. We detail methods of the workflow from ... [摘要]  将寡核苷酸治疗剂即siRNAs有效递送到中枢神经系统代表了治疗神经系统疾病的临床进展的显着障碍。 小的内源性细胞外囊泡显示能够在细胞(包括神经元)之间传输脂质,蛋白质和RNA。 这种天然的贩运能力使细胞外囊泡成为寡核苷酸即siRNA的递送载体的潜力。 然而,缺乏用寡核苷酸载体装载细胞外囊泡的稳健和可扩展的方法。 我们描述了在简单共孵育后将疏水修饰的siRNA加载到细胞外囊泡中的详细方案。 我们详细介绍了从细胞外囊泡纯化到数据分析的工作流程。 该方法可以促进基于细胞外基于囊泡的疗法用于治疗广泛的神经障碍。
【背景】siRNA是一种类型的寡核苷酸治疗剂,一类新的直接靶向信使RNA(mRNA)的药物,以防止导致疾病表型的蛋白质的表达。 siRNA的治疗应用是非常有希望的,因为siRNA可以被设计为靶向任何基因,包括不能用小分子或基于蛋白质的疗法“可药用”的基因。在寡核苷酸治疗剂的化学中取得的进展使得能够设计完全稳定的疏水改性的siRNA(hsiRNA,用2'-O-甲基或2'-氟以及硫代磷酸酯和共价键合到胆固醇的有义链修饰),其促进细胞hsiRNA的自我内化,并保持有效负载在RNA诱导的沉默复合体(RISC)中的能力(Byrne等,2013; Khvorova和Watts,2017)。与乘客链的3'末端连接的胆固醇缀合物对于快速细胞膜缔合是必需的(Byrne等,2013; ...

Lipid Extraction from HeLa Cells, Quantification of Lipids, Formation of Large Unilamellar Vesicles (LUVs) by Extrusion and in vitro Protein-lipid Binding Assays, Analysis of the Incubation Product by Transmission Electron Microscopy (TEM) and by Flotation across a Discontinuous Sucrose Gradient
Author:
Date:
2016-10-20
[Abstract]  Dissecting the interactions established between proteins and membranes in a given type of cells is not an easy task. Using a cell-free system of large unilamellar vesicles (LUVs) to analyze these interactions may help decipher these interactions and identify potential membrane deformations induced by the proteins incubated with these LUVs. This article describes the protocols for 1) extraction of total lipids from eukaryotic cells using the method developed by Bligh and Dyer (1959), 2) the quantification of glycerophospholipids by gas chromatography after methanolysis, followed by 3) the formation of LUVs by extrusion, 4) protein-lipid binding assay, 5) analysis of the incubation product by transmission electron microscopy (TEM) and by flotation across a discontinuous sucrose gradient and ... [摘要]  解剖在给定类型的细胞中蛋白质和膜之间建立的相互作用不是一个容易的任务。使用大单层囊泡(LUV)的无细胞系统来分析这些相互作用可以帮助破译这些相互作用和识别由与这些LUV孵育的蛋白质诱导的潜在的膜变形。本文介绍了1)使用由Bligh和Dyer(1959)开发的方法从真核细胞中提取总脂质,2)在甲醇分解后通过气相色谱法定量甘油磷脂,然后3)通过挤出形成LUV的方案, 4)蛋白质 - 脂质结合测定,5)通过透射电子显微镜(TEM)和通过不连续蔗糖梯度浮选分析孵育产物,最后,6)通过免疫印迹分析蛋白质并通过碘素熏蒸显示甘油磷脂。

[背景] 包含巨单层囊泡(GUV;由单个磷脂双层组成,直径大于1μm)或脂质体孵育的无细胞系统与重组蛋白可能有助于了解这些相互作用。根据它们的直径和层数,脂质体被分为小的单层囊泡(SUV;由单个磷脂双层构成的囊泡,直径在20和100nm之间),大的单层囊泡(LUV;由单个双层磷脂,并且直径在100和400nm之间),大多层囊泡(MLV;由多个磷脂双层构成且直径在200nm和3μm之间的囊泡)和多泡囊泡(MVV);由囊泡组成的大囊泡单个双层磷脂,并含有几个较小的囊泡,每个囊泡由单个双层磷脂组成)。 ...

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