Name: preparation of embryos for electron microscopy of the drosophila embryonic heart tube
Uploaded: 2009-12-21T10:00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube at JoVE.com

我们的心管形成的工作是支持由美国国家科学基金会的资助（奖励编号0744165）SGK

<ol><li>刚准备一个含有20毫升的玻璃闪烁瓶12.5％戊二醛在50MM Cacodylate缓冲液（pH7.4）固定液的解决方案2毫升。加入正庚烷8毫升大力动摇。允许分开的两个阶段。到一个干净的闪烁瓶中取出上相含戊二醛饱和的正庚烷，地点和预留。这将是固定液。 </li><li>收集0-20小时的胚胎，利用胚胎收集室，这是一个可移动的网格插入的小篮子。 50％的漂白粉溶液中浸泡2-3分钟胚胎取出的外蛋壳膜，或直到胚​​胎浮到表面的漂白剂。彻底冲洗用纸巾上ddH20和杂交胚胎。 </li><li>使用镊子，拿起覆盖dechorionated胚胎和成的固定液，使胚胎下降网格的网格插入。允许胚胎修正为1.5小时，在4 ° C，而在一个Nutator混合。 </li><li>准备一个培养皿内衬双面胶带。从固定液使用巴斯德吸管取出胚胎和他们放到录音的Petri dish.Transfer表面少量的胚胎在固定液固定液庚，以防止溶解的磁带的粘合剂。摇动培养皿中的胚胎单层。直到庚蒸发的防护罩放置在培养皿。加入足够的PBS + 0.1％Tween20到胚胎。手devitellinize后期阶段的16个胚胎解剖显微镜下用钝器钨针。恢复到1.5 ml离心管巴斯德吸管的胚胎。在此离心管进行步骤5-7。 </li><li>冲洗胚胎0.1M Cacodylate缓冲液，pH 7.4，然后在0.1M Cacodylate缓冲液，pH 7.4在室温下1小时，含1％四氧化锇溶液后修复。冲洗0.1M Cacodylate缓冲液，pH值7.4的胚胎。 </li><li>在乙醇和丙酮梯度脱水胚胎。胚胎脱水用50％乙醇10分钟，70％乙醇10分钟。然后在90％丙酮脱水10分钟，和两个100％丙酮，每次10分钟的步骤。 </li><li>删除丙酮，然后开始加入1:1的EPON Spurr树脂渗透的过程：丙酮混合胚胎。微波三分钟使用Pelco的功能弹性临微波真空压力下，同时保持样品。申请一个额外的5分钟后，已经结束的微波真空压力的样品。交易所1:1树脂，丙酮100％EPON - Spurr树脂和微波真空压力下。交易所的100％EPON - Spurr树脂最后的时间，和微波真空压力下。 </li><li>使用巴斯德吸管，胚胎转移的离心管，以硅树脂嵌入模具。成一排，这样的胚胎后结束与模具的锥形边缘对齐，对齐胚胎。烘烤在70 ° C烘箱过夜。 </li><li>取出样品，准备切片。 </li><li>开始切割1μm的部分，用金刚石HISTO刀和里克特Ultracut发送切片。请注意，当第一个胚胎部分达到。从这一点来说，削减100μm的进样。删除部分使用循环，并到载玻片的地方。简要地热的一个热点板块的部分。使用一滴亚甲蓝染色部分。在光学显微镜下识别心腔。 </li><li>切割使用90纳米钻石超45 °刀和里克特Ultracut发送切片部分。拿起一个铜栅上的多个部分。 </li><li>网格3％的铀，50％的乙醇10分钟饱和醋酸染色，然后在双蒸水冲洗三次。然后，电网染色柠檬酸铅中含有氢氧化钠颗粒创建一个CO 2，自由的环境室（中10毫升双蒸水和100ml 10M NaOH溶液溶解0.04gms）为2.5分钟。电网双蒸水冲洗三次。 </li><li>节用JEOL 1200EX电子显微镜检查，在80KV，AMT数码相机拍摄的。 </li></ol>

<ol>
	<li>Tepass, U., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Development+of+Cellular+Junctions+in+the+Drosophila+embryo.">The Development of Cellular Junctions in the Drosophila embryo.</a> Dev. Biol. 161, 563-596 (1994).</li><li>Sullivan, W., Ashburner, M., Hawley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila Protocols. , (2000).</li><li>Santiago-Martinez, E., Soplop, N. H., Patel, R., Kramer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repulsion+by+Slit+and+Roundabout+prevents+Shotgun/E-cadherin-mediated+cell+adhesion+during+Drosophila+heart+tube+lumen+formation.">Repulsion by Slit and Roundabout prevents Shotgun/E-cadherin-mediated cell adhesion during Drosophila heart tube lumen formation.</a> J. Cell Biol. 182, 241-248 (2008).</li></ol>

果蝇胚胎的心管的形态发生，已成为一个有价值的模型系统，为研究胚胎发育过程中的细胞粘附和细胞形态的变化。在研究这种结构所面临的挑战之一是，很难想象在整装胚胎的心管的管腔。在这个视频演示的技术，这是从前面描述的程序1-2适应，已被证明是有效和可靠地分析大量的基因型心管和管腔形成3的成功。

preparation of embryos for electron microscopy of the drosophila embryonic heart tube

的形态发生<em&gt;果蝇</em&gt;胚胎心管已成为一个有价值的模型系统，为研究胚胎发育过程中细胞迁移，细胞与细胞粘附和细胞形态的变化。研究这种结构所面临的挑战之一，就是心管的管腔，以及膜的功能，心管的形成是至关重要的，很难想象在整装胚胎，由于体积小，心管和内部lumenal的空间相对胚胎。透射电子显微镜的使用，使这些结构的放大倍率较高，并给出了截面，这就容易揭示管腔的大小和形状的胚胎研究的优势。在这段视频中，我们详细可靠的固定的过程中，嵌入，和后期切片<em&gt;果蝇</em&gt;胚胎，以形象化的心脏管腔以及重要的细胞结构，包括细胞与细胞间的路口和基底膜。

Watch this Scientific Journal Video about Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube at JoVE.com

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

我们描述了一个固定，包埋过程，切片，和后期成像<em&gt;果蝇</em&gt; Trasmission电子显微镜对胚胎的心管的胚胎。这种技术可以让心脏管腔的可视化以及基底膜，哪条线路的心腔。

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Research

JoVE Journal

Biology

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

科研

生物学

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

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Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

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The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

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可视化心脏跳动果蝇</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

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The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

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Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

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Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

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Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

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The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...