Optic Nerve Crush in Mice to Study Retinal Ganglion Cell Survival and Regeneration
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Author:
Date:
2020-03-20
[Abstract] In diseases such as glaucoma, the failure of retinal ganglion cell (RGC) neurons to survive or regenerate their optic nerve axons underlies partial and, in some cases, complete vision loss. Optic nerve crush (ONC) serves as a useful model not only of traumatic optic neuropathy but also of glaucomatous injury, as it similarly induces RGC cell death and degeneration. Intravitreal injection of adeno-associated virus serotype 2 (AAV2) has been shown to specifically and efficiently transduce RGCs in vivo and has thus been proposed as an effective means of gene delivery for the treatment of glaucoma. Indeed, we and others routinely use AAV2 to study the mechanisms that promote neuroprotection and axon regeneration in RGCs following ONC. Herein, we describe a step-by-step protocol to ...
[摘要] [摘要 ] 在青光眼等疾病中,视网膜神经节细胞(RGC)神经元无法存活或无法再生视神经轴突,这是部分视力丧失的原因,在某些情况下,甚至是完全的视力丧失。视神经挤压术(ONC)不仅可以作为创伤性视神经病变的一种有用模型,而且还可以作为青光眼损伤的有用模型,因为它类似地诱导RGC细胞死亡和变性。腺相关病毒血清型2(AAV2)的玻璃体内注射已被证明特别地和有效地转导视网膜神经节细胞在体内和已因而被提出作为基因递送用于治疗青光眼的治疗的有效手段。确实,我们和其他人常规使用AAV2来研究促进ONC 后RGC中神经保护和轴突再生的机制。本文中,我们描述了分步操作的方案,以测定AAV2介导的转导和ONC损伤后小鼠中RGC的存活和再生,包括1)玻璃体内注射AAV2病毒载体,2)视神经挤压,3)霍乱毒素B (CTB)标记再生轴突,4)视神经清除,5)视网膜平面免疫染色和6)定量RGC存活和再生。除了提供执行此协议所需的所有材料和程序详细信息之外,我们还强调了它比其他相似的已发表方法的优势,并提供了有用的技巧以确保其在任何现代实验室中都能如实复制。
[背景 ] 青光眼是世界范围内不可逆失明的主要原因,其特征是视网膜神经节细胞(RGCs)逐渐退化和丧失,这是构成连接视网膜与大脑的视神经的中央投射神经元(Quigley ,2011 ; Tham ...
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Structural Analysis of Bordetella pertussis Biofilms by Confocal Laser Scanning Microscopy
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Author:
Date:
2018-08-05
[Abstract] Biofilms are sessile communities of microbial cells embedded in a self-produced or host-derived exopolymeric matrix. Biofilms can both be beneficial or detrimental depending on the surface. Compared to their planktonic counterparts, biofilm cells display enhanced resistance to killing by environmental threats, chemicals, antimicrobials and host immune defenses. When in biofilms, the microbial cells interact with each other and with the surface to develop architecturally complex multi-dimensional structures. Numerous imaging techniques and tools are currently available for architectural analyses of biofilm communities. This allows examination of biofilm development through acquisition of three-dimensional images that can render structural features of the sessile community. A frequently ...
[摘要] 生物膜是嵌入自生或宿主衍生的外聚合物基质中的微生物细胞的固着群落。根据表面,生物膜可以是有益的或有害的。与浮游生物相比,生物膜细胞表现出更强的抗环境威胁,化学物质,抗菌药物和宿主免疫防御能力。当处于生物膜中时,微生物细胞彼此相互作用并与表面相互作用以形成结构复杂的多维结构。目前,许多成像技术和工具可用于生物膜群落的建筑分析。这允许通过获取可以呈现无柄群落的结构特征的三维图像来检查生物膜的发展。经常使用的工具是共聚焦激光扫描显微镜。我们提出了一个详细的协议,以生长,观察和分析呼吸道人类病原体,百日咳博德特氏菌的生物膜在空间和时间。
【背景】百日咳博德特氏菌(Bordetella pertussis)是上呼吸道的专性人类病原体,引起百日咳或百日咳(Mooi,2010; Dorji et al。,2018)。 B的生物膜。百日咳在各种人造表面上以及静态,摇动和流体流动条件下形成(Mishra et al。,2005; Sloan et al。,2007 ; Serra et al。,2011)。对这些生物膜的显微评估表明,这种细菌产生不规则形状的微集落,由流体通道分隔,嵌入由细胞外DNA(eDNA),蛋白质和多糖组成的外聚合物基质中(Parise et al。,2007; Sloan et al。,2007; Serra et al。,2008; ...
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Centromere Chromosome Orientation Fluorescent in situ Hybridization (Cen-CO-FISH) Detects Sister Chromatid Exchange at the Centromere in Human Cells
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Author:
Date:
2018-04-05
[Abstract] Human centromeres are composed of large tandem arrays of repetitive alpha satellite DNA, which are often sites of aberrant rearrangement in cancers (Mitelman et al., 1997; Padilla-Nash et al., 2001). To date, annotation of the human centromere repetitive sequences remains incomplete, greatly hindering in-depth functional studies of these regions essential for chromosome segregation. In order to monitor sister chromatid exchange happening at the centromere (C-SCE) due to recombination and mutagenic events, I have applied the Chromosome-Orientation Fluorescence in situ Hybridization (CO-FISH) technique to centromeres (Cen-CO-FISH) in human cells. This hybridization-based method involves (1) the incorporation of nucleotide analogs through a single round of ...
[摘要] 人类着丝粒由重复的α卫星DNA的大串联阵列组成,这些细胞通常是癌症中异常重排的位点(Mitelman等人,1997; Padilla-Nash等人 >,2001)。迄今为止,对人类着丝粒重复序列的注释仍然不完整,极大地妨碍了这些区域对染色体分离至关重要的深入功能研究。为了监测由于重组和诱变事件而在着丝粒(C-SCE)上发生姊妹染色单体交换,我将染色体定位荧光原位杂交(CO-FISH)技术应用于着丝粒( Cen-CO-FISH)在人类细胞中的表达。这种基于杂交的方法包括(1)通过单轮复制掺入核苷酸类似物,(2)新合成的DNA链的酶消化和(3)单链探针的后续杂交,在不存在变性步骤的情况下。所产生的信号允许基于DNA的5'-3'方向性差异地标记每个姊妹染色单体,并评估指示C-SCE的异常染色模式。应用于人类着丝粒的Cen-CO-FISH方法揭示,人类着丝粒确实在循环细胞中发生重组,导致C-SCE,并且在经历衰老的癌细胞系和原代细胞中着丝粒不稳定性增强(Giunta和Funabiki,2017)。在这里,我介绍了人类细胞中Cen-CO-FISH方法的制备,实验程序和数据采集的详细方案。它还包括该技术的概念性概述,以及代表性图像和评分准则的示例。 Cen-CO-FISH是促进着丝粒重复探索的有用工具。
【背景】人类基因组计划于2003年标记为完成,但它遗漏了超过10%的人类重复DNA(de ...
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