Name: immunostaining of dissected zebrafish embryonic heart
Uploaded: 2012-01-10T12:00:00
Description: Watch this Scientific Journal Video about Immunostaining of Dissected Zebrafish Embryonic Heart at JoVE.com

我们感谢他的帮助在斑马鱼的饲养Beninio Jomok。这项工作是由国立卫生研究院HL81753资助。

1。幻灯片的制备和湿盒<ol><li>湿盒，可从一个盒子为空的提示框等。包装室和用铝箔盖，以保护样品从光。 </li><li>箱内，放了一堆湿纸巾保持室内的湿度，防止干燥的心脏样本。设置上的纸毛巾作为幻灯片的机架顶部的小微孔板。 </li><li>在表面的显微镜幻灯片使用immEdge笔关于采用5x5 mm的尺寸与心脏样本井要么绘制线或圆形。让幻灯片干燥。 </li><li>湿盒中的幻灯片，并添加50ul新鲜的4％，每口?...

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	<li>Shin, J. T., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Zebrafish+to+human:+modular+medical+models.">From Zebrafish to human: modular medical models.</a> Annu. Rev. Genomics Hum. Genet. 3, 311-340 (2002).</li><li>Beis, D., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cell+biology:+following+the+zebrafish+trend.">In vivo cell biology: following the zebrafish trend.</a> Trends. Cell. Biol. 16, 105-112 (2006).</li><li>Schoenebeck, J. J., Yelon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+cardiac+development:+Advances+in+imaging+add+new+dimensions+to+the+utility+of+zebrafish+genetics.">Illuminating cardiac development: Advances in imaging add new dimensions to the utility of zebrafish genetics.</a> Semin. Cell. Dev. Biol. 18, 27-35 (2007).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nat. Genet. 26, 216-220 (2000).</li><li>Dahme, T., Katus, H. A., Rottbauer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fishing+for+the+genetic+basis+of+cardiovascular+disease.">Fishing for the genetic basis of cardiovascular disease.</a> Dis. Model Mech. 2, 18-22 (2009).</li><li>Huang, W., Zhang, R., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibrillogenesis+in+the+developing+zebrafish+heart:+A+functional+study+of+tnnt2.">Myofibrillogenesis in the developing zebrafish heart: A functional study of tnnt2.</a> Dev. Biol. 331, 237-249 (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book. , (2007).</li><li>Buckingham, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle:+the+regulation+of+myogenesis.">Muscle: the regulation of myogenesis.</a> Curr. Opin. Genet. Dev. 4, 745-751 (1994).</li><li>Wheelock, M. J., Knudsen, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherins+and+associated+proteins.">Cadherins and associated proteins.</a> Vivo. 5, 505-513 (1991).</li><li>Sun, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+hypertrophy+involves+both+myocyte+hypertrophy+and+hyperplasia+in+anemic+zebrafish.">Cardiac hypertrophy involves both myocyte hypertrophy and hyperplasia in anemic zebrafish.</a> PLoS One. 4, e6596-e6596 (2009).</li><li>Auman, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+modulation+of+cardiac+form+through+regionally+confined+cell+shape+changes.">Functional modulation of cardiac form through regionally confined cell shape changes.</a> PLoS Biol. 5, e53-e53 (2007).</li><li>Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+zebrafish+essential+and+regulatory+myosin+light+chains+reduces+cardiac+function+through+distinct+mechanisms.">Depletion of zebrafish essential and regulatory myosin light chains reduces cardiac function through distinct mechanisms.</a> Cardiovasc. Res. 79, 97-108 (2008).</li></ol>

我们的方法，以经典的整装免疫组化方法相比，具有以下优点。首先，可以始终获得更强的荧光信号由于提高普及率。在整个装载免疫方法，密集的皮肤组织周围的心脏显著减少许多抗体的渗透，在高背景导致整个身体。这个问题严重，特别是对超过3天受精后（DPF）的胚胎。相比之下，解剖心中只由几个细胞层，呈现更好的渗透。其次，由于已大为改善渗透，全过程的免疫可以减少从1天到只有?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> tricaine </td><td>研究有机物</td><td> 3007T </td></tr><tr><td>甲醛</td><td> Polysciences </td><td> 04018 </td></tr><tr><td>胰岛素注射器</td><td>碧迪</td><td> 329461 </td></tr><tr><td> TRITON - X - 100 </td><td>西格玛</td><td> T8532 </td></tr><tr><td>抗β- catenin抗体</td><td>西格玛</td><td> C7207 </td></tr><tr><td>反MEF2C抗体</td><td>圣克鲁斯生物技术</td><td> SC313 </td></tr><tr><td> F59 </td><td> ZFIN </td><td></td></tr><tr><td>抗-α-辅肌动蛋白抗体</td><td>西格玛</td><td> A7811 </td></tr><tr><td>反ILK抗体</td><td>细胞信号转导</td><td> ＃3862 </td></tr><tr><td>的Alexa Fluor 568山羊抗兔IgG </<html> TD&gt; <td> Invitrogen公司</td><td> A11011 </td></tr><tr><td>的Alexa Fluor 488山羊抗鼠IgG1 </td><td> Invitrogen公司</td><td> A21121 </td></tr><tr><td>荧光的安装介质</td><td>矢量</td><td> H - 1200 </td></tr><tr><td> ImmEdge笔</td><td>矢量</td><td> H - 4000 </td></tr><tr><td>聚- L -赖氨酸涂层幻灯片</td><td>电子显微镜科学</td><td> 63410-01 </td></tr><tr><td>显微镜盖玻片</td><td>费舍尔</td><td> 12 - 543 - D </td></tr><tr><td>凹形显微镜幻灯片</td><td>费舍尔</td><td> 7-1305-8 </td></tr><tr><td>解剖镜下</td><td>徕卡MZ95 </td><td></td></tr><tr><td>复式显微镜</td><td>蔡司</td><td> Axioplan2 </td></tr><tr><td> ApoTome </td><td>蔡司</td><td></td></tr></tbody></table> PBST： 1 × PBS 0.5％的Triton X - 100 耳鼻喉科“&gt; 25X Tricaine： 400毫克Tricaine 97.9毫升DDH 2 O 2.1毫升（1M三PH9） 调整pH值至7.0

immunostaining of dissected zebrafish embryonic heart

斑马鱼的胚胎成为在体内的脊椎动物模型研究心脏发育和人类心脏疾病，由于其优越的胚胎学和遗传学1,2的流行。约100-200胚胎都是现成的，每周从一对成鱼。开发前子宫内的透明胚胎，使他们理想的评估心脏缺陷3。通过吗啉技术或RNA注射4的任何基因的表达可以被操纵。此外，遗传筛选已经产生的突变体，影响不同的角度cardiogenesis 5 。 整个装载免疫组化是一个重要的技术在这种动物模型揭示的目标蛋白质的表达模式，以一个特定的组织 6 。然而，高清晰度的图像，可以揭示细胞或亚细胞结构已经很难，主要是由于物理locat离子的心脏和较差的抗体渗透。 在这里，我们目前通过解剖心脏载玻片表面上，然后再进行染色过程中的一个方法来解决这些瓶颈。为了防止小心脏样品的损失，并促进解决方案的处理，我们限制在一个圆圈上immEdge笔绘制表面的显微镜幻灯片心脏样本。染色后，可直接观察到的荧光信号的复合显微镜。 我们的新方法显着提高抗体的渗透，因为从一个胚胎的鱼心脏只有几个细胞层组成。年龄从2天到6天的斑马鱼胚胎内大幅减少的游行时间从完整的心可以得到高质量的图像。我们的方法可以潜在地扩展到染色无论从斑马鱼或其他小动物解剖的其他器官。

Watch this Scientific Journal Video about Immunostaining of Dissected Zebrafish Embryonic Heart at JoVE.com

Immunostaining of Dissected Zebrafish Embryonic Heart

一个快速的方式来进行免疫的斑马鱼胚胎心脏描述。整装免疫方法相比，这种方法大大增加渗透的抗体，这使得获取高分辨率图像，揭示了心脏细胞/亚细胞结构内大幅减少处理时间。

免疫染色的解剖斑马鱼胚胎心脏

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Research

JoVE Journal

Biology

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

科研

生物学

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

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

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

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 ...

Microdissection of Zebrafish Embryonic Eye Tissues

斑马鱼胚胎眼组织显微切割的

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

全安装胚胎肢体皮肤血管的免疫组织化学分析：一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

A System for ex vivo Culturing of Embryonic Pancreas

A System for ex vivo Culturing of Embryonic Pancreas

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

激光伤害的斑马鱼胚胎骨骼肌

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

从解离分析准备胚胎和幼体斑马鱼骨肌纤维

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

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