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TritonTM X-100

Triton TM X-100

Company: Sigma-Aldrich
Catalog#: T9284
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Dissection and Whole Mount Staining of Retina from Neonatal Mice
Author:
Date:
2018-10-05
[Abstract]  Here we provide a detailed protocol for whole mount staining of mouse retina. This protocol was used to analyze retinal angiogenesis in newborn mice (Sawaguchi et al., 2017) by modifying the original protocols (Powner et al., 2012; Tual-Chalot et al., 2013). This protocol can also be used for whole mount staining of adult retina. [摘要]  在这里,我们提供了小鼠视网膜整体染色的详细方案。 该方案用于分析新生小鼠的视网膜血管生成(Sawaguchi et al。,2017),修改原始方案(Powner et al。,2012; Tual-Chalot et al。,2013)。 该方案也可用于成人视网膜的整体染色。

Preserve Cultured Cell Cytonemes through a Modified Electron Microscopy Fixation
Author:
Date:
2018-07-05
[Abstract]  Immunocytochemistry of cultured cells is a common and effective technique for determining compositions and localizations of proteins within cellular structures. However, traditional cultured cell fixation and staining protocols are not effective in preserving cultured cell cytonemes, long specialized filopodia that are dedicated to morphogen transport. As a result, limited mechanistic interrogation has been performed to assess their regulation. We developed a fixation protocol for cultured cells that preserves cytonemes, which allows for immunofluorescent analysis of endogenous and over-expressed proteins localizing to the delicate cellular structures. [摘要]  培养细胞的免疫细胞化学是用于确定细胞结构内蛋白质的组成和定位的常用且有效的技术。 然而,传统的培养细胞固定和染色方案不能有效地保存培养的细胞色素,长期专门用于形态发生转运的丝状伪足。 结果,进行了有限的机械审讯以评估其监管。 我们开发了一种用于培养细胞的固定方案,该方案保留了细胞质,允许对内源性和过表达的蛋白质进行免疫荧光分析,这些蛋白质定位于脆弱的细胞结构。

【背景】Cytonemes被分类为薄的(~200nm直径)基于肌动蛋白的丝状伪足,长度超过2μm,可以转运形态发生素(Ramírez-Weber和Kornberg,1999)。这些信号结构首先在发育中的 Drosophila 翼成像盘中进行了详细分类和描述,随后在小鼠,小鸡和斑马鱼模型生物中进行了观察(Ramírez-Weber和Kornberg,1999; Sanders et al。,2013; Stanganello et al。,2015)。在大多数情况下,只有对过表达的荧光标记蛋白进行实时成像才能进行细胞色素检测。由于传统的固定方案未能保存这些脆弱的细丝,因此对培养细胞的细胞色素的检查受到限制。这些并发症一直是决定在发育和组织稳态期间驱动细胞色素形成和功能的细胞机制以及确定这些过程是否在疾病中被破坏的限制因素。

为了克服这些限制,我们开发了一种基于修饰电子显微镜固定剂(MEM-fix)的方案,该方案可以保留培养细胞的细胞质。 ...

Detecting the Interaction of Double-stranded RNA Binding Protein, Viral Protein and Primary miRNA Transcript by Co-immunoprecipitation in planta
Author:
Date:
2018-05-05
[Abstract]  MicroRNAs (miRNAs) play important roles in plant growth, development, and response to infection by microbes. Double-stranded RNA binding protein 1 (DRB1) facilitates the processing of primary miRNA transcripts into mature miRNAs. Recently, we found that NS3 protein encoded by rice stripe virus (RSV) associates with DRB1 and promotes miRNA biogenesis during RSV infection (Zheng et al., 2017). RNA co-immunoprecipitation (RIP) method was applied to identity association patterns among DRB1, NS3, and miRNA transcript. [摘要]  微小RNA(miRNA)在植物生长,发育和微生物感染反应中发挥重要作用。 双链RNA结合蛋白1(DRB1)有助于将初级miRNA转录物加工成成熟的miRNA。 最近,我们发现水稻条纹病毒(RSV)编码的NS3蛋白与DRB1相关并促进RSV感染期间的miRNA生物合成(Zheng等人,2017)。 RNA共免疫沉淀(RIP)方法被用于鉴定DRB1,NS3和miRNA转录物之间的关联模式。

【背景】在双链RNA(dsRNA)结合蛋白HYPONASTIC LEAVES1(DRB1 / HYL1)的帮助下,通过RNA酶III酶DICER-LIKE1(DCL1)从其初级转录物(pri-miRNA) 锌指蛋白SERRATE(SE)。 水稻条纹病毒(RSV)感染广泛地干扰miRNA积累。 我们发现RSV编码的非结构蛋白3(NS3)通过与水稻中的DRB1相互作用下调pri-miRNAs来促进miRNA积累(Zheng等人,2017)。 为了揭示NS3如何增强pri-miRNA的加工,我们使用免疫共沉淀(Co-IP)来说明NS3,DRB1和pri-miRNA体内的关系。 该协议有助于了解两种蛋白质和一种RNA转录本之间的关联模式。

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