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Glutaraldehyde solution 25%

戊二醛溶液

Company: Sigma-Aldrich
Catalog#: G5882
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Imaging Microtubules in vitro at High Resolution while Preserving their Structure
Author:
Date:
2021-04-05
[Abstract]  

Microtubules (MT) are the most rigid component of the cytoskeleton. Nevertheless, they often appear highly curved in the cellular context and the mechanisms governing their overall shape are poorly understood. Currently, in vitro microtubule analysis relies primarily on electron microscopy for its high resolution and Total Internal Reflection Fluorescence (TIRF) microscopy for its ability to image live fluorescently-labelled microtubules and associated proteins. For three-dimensional analyses of microtubules with micrometer curvatures, we have developed an assay in which MTs are polymerized in vitro from MT seeds adhered to a glass slide in a manner similar to conventional TIRF microscopy protocols. Free fluorescent molecules are removed and the MTs are fixed by perfusion. The MTs can

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[摘要]  [摘要]微管(MT)是细胞骨架中最刚性的组成部分。然而,它们在细胞环境中经常显得高度弯曲,并且控制它们整体形状的机理了解甚少。当前,体外微管分析主要依靠电子显微镜进行高分辨率分析,而全内反射荧光(TIRF )显微镜则可以对活的荧光标记的微管和相关蛋白进行成像。为了对具有微米曲率的微管进行三维分析,我们开发了一种在体外聚合MT的检测方法 用类似于常规TIRF显微镜操作规程的方式将MT种子的MT粘附到载玻片上。除去游离的荧光分子,并通过灌注固定MTs。然后可以使用带有Airyscan模块的共聚焦显微镜观察MT,以获得更高的分辨率。该协议允许对保留其原始三维形状并与高分辨率免疫荧光检测兼容的微管进行成像。

[背景]微管(MT)是通过异源二聚体的组合制成的聚合物α和β微管蛋白,并且是细胞骨架的主要成分。他们参与了细胞功能的基本机制,如有丝分裂,细胞内转运,胞质分裂和细胞形态的维持(Akhmanova和Steinmetz,2015)。尽管MT本身具有很高的刚性,但它们通常会在细胞中弯曲并产生一些蛋白,从而弯曲微管(Brangwynne等人,2006; Bechstedt等人,2014; Leung等人,2020; Cuveillier等人,2020 ...

Molecular and Phenotypic Characterization Following RNAi Mediated Knockdown in Drosophila
Author:
Date:
2021-02-20
[Abstract]  

Loss of function studies shed significant light on the involvement of a gene or gene product in different cellular processes. Short hairpin RNA (shRNA) mediated RNA interference (RNAi) is a classical yet straightforward technique frequently used to knock down a gene for assessing its function. Similar perturbations in gene expression can be achieved by siRNA, microRNA, or CRISPR-Cas9 methods also. In Drosophila genetics, the UAS-GAL4 system is utilized to express RNAi and make ubiquitous and tissue-specific knockdowns possible. The UAS-GAL4 system borrows genetic components of S. cerevisiae, hence rule out the possibility of accidental expression of the system. In particular, this technique uses a target-specific shRNA, and the expression of the same is governed by the upstream activating

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[摘要]  [摘要]功能丧失的研究为基因或基因产物在不同细胞过程中的参与提供了重要启示。短发夹RNA(shRNA)介导的RNA干扰(RNAi)是一种经典而直接的技术,经常用于敲低基因以评估其功能。也可以通过siRNA,microRNA或CRISPR-Cas9方法实现类似的基因表达扰动。在果蝇遗传学中,UAS-GAL4系统用于表达RNAi,并使遍在和组织特异性的基因敲除成为可能。UAS-GAL4系统借鉴了酿酒酵母的遗传成分,因此排除了系统意外表达的可能性。特别地,该技术使用靶标特异性shRNA,并且其表达受上游激活序列(UAS)支配。由特定启动子调节的GAL4受控表达可以普遍或以组织特异性方式驱动干扰RNA的表达。通过RNA分离和半定量RT-PCR反应,然后进行琼脂糖凝胶电泳来测量敲低效率。我们还采用了免疫染色程序来评估击倒效率。

RNAi为研究人员提供了降低基因产物水平(相当于亚同型条件)并研究结果的选择。基于UAS-GAL4的RNAi方法提供了基因表达的时空调节,还有助于推断早期发育阶段所需的基因功能。


[背景]果蝇果蝇(果蝇)是在研究实验室经常使用的一种通用模式生物。果蝇易于处理,繁殖和维护。而且,精心制作却寿命短,繁殖力高的果蝇具有更多的优势。果蝇遗传学工具的易用性有助于发展对基因功能的全面了解。由于果蝇基因中有60%与人类基因同源,并且具有前面提到的其他优点,因此果蝇是研究体内基因功能的显而易见的模型生物。 ...

Nematode Epicuticle Visualisation by PeakForce Tapping Atomic Force Microscopy
Author:
Date:
2017-11-05
[Abstract]  The free-living soil nematode Caenorhabditis elegans has become an iconic experimental model animal in biology. This transparent animal can be easily imaged using optical microscopy to visualise its organs, tissues, single cells and subcellular events. The epicuticle of C. elegans nematodes has been studied at nanoscale using transmission and scanning (SEM) electron microscopies. As a result, imaging artefacts can appear due to embedding the worms into resins or coating the worms with a conductive gold layer. In addition, fixation and contrasting may also damage the cuticle. Conventional tapping mode atomic force microscopy (AFM) can be applied to image the cuticle of the dried nematodes in air, however this approach also suffers from imaging defects. Ideally, the ... [摘要]  自由生活的土壤线虫秀丽隐杆线虫已经成为生物学中一个标志性的实验动物模型。这种透明的动物可以很容易地使用光学显微镜成像,以可视化其器官,组织,单细胞和亚细胞事件。 C的角质层。已经使用透射和扫描(SEM)电子显微镜在纳米级研究了线虫线虫。结果,由于将蠕虫嵌入树脂或用导电金层涂覆蠕虫,可能出现成像伪像。另外,固定和对比也可能损伤角质层。传统的敲击模式原子力显微镜(AFM)可用于对干燥的线虫的角质层在空气中成像,然而这种方法也存在成像缺陷。理想情况下,线虫应该在类似自然环境的条件下成像。最近,我们报道了使用PeakForce攻丝AFM模式来成功实现可视化和数值表征。线虫角质层在空气和液体中均有表达(Fakhrullina et al。,2017)。我们成像的主要线虫表面结构和角质层的力学性能表征。该协议提供了对液体浸泡的AF的AFM成像的详细描述。线虫使用PeakForce攻丝原子力显微镜线虫。
【背景】自由生活和寄生虫的线虫由于其对农业和人类福祉的显着生物学效应而受到广泛的研究。显然,线虫中研究最多和最着名的物种是自由生活的土壤线虫秀丽隐杆线虫(Sterken等人,2015年)。这种蠕虫在许多研究中已被成功地用作多功能模式生物体(Fire等人,1998; Kenyon,2010; Swierczek等人,2011; O'Reilly等人,2014; ...

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