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％，每口井的甲醛。 </li></ol> 2。胚胎心脏解剖<ol><li>在E3鸡蛋的水中含有约1分钟，直至鱼停止身体运动0.4％tricaine麻醉鱼胚胎，但仍然有正常的心脏跳动。 </li><li>凹显微镜幻灯片放在一个解剖镜下阶段。凹形以及胚胎转移。 </li><li>调整清楚地揭示了心脏的解剖显微镜的光组合。根据个人喜好，它可以是暗场或DIC样偏振光条件。 </li><li>两个胰岛素注射器用75％乙醇的清洁，然后晾干。 </li><li>解剖镜下调整到更高的放大倍率和心脏的重点。身体修复的胚胎，卵黄体节的边界，接近头部插入一个针头。插入另一针靠近心脏地区，并迅速拉心脏。如果心脏是没有完全从胚胎中分离出来，切断连接心脏和蛋黄之间的剩余组织。 </li><li>调整显微镜低倍率和分离心脏的焦点。使用加入10μlpipetteman，将其设置为加入1μl体积。应用pipetteman的一角，可以借鉴或触摸心脏样本，使离体心脏内或针尖表面。 </li><li>浸在准备幻灯片4％的甲醛溶液中的吸头和压低吸管活塞释放到溶液中心脏。水的表面张力也有助于释放的心脏从枪头的解决方案。少装比3，在每口井的心中。 </li></ol> 3。免疫染色<ol><li>关闭室，并放在一个振动筛用温和的摇摆运动。修复心脏样品在室温下20分钟。 </li><li>后面的幻灯片上的水擦去，并把幻灯片上的一个缓冲交换的解剖镜下阶段。 </li><li>浸在每口井从加入10μlpipetteman到一个空地区的一角，并尽可能保持心脏样品的提示。采取警告，以避免心脏附加给小费，因为心可能与水流移动时的解决办法是绘制成尖。更改提示本地化，如果需要的话。 </li><li>处置到一个组织的解决方案，避免产生任何气泡，在尖，因为心脏样本很容易失去气泡的存在。保持心脏样本部分干很短的时间，可以帮助保持连接到幻灯片的心。 </li><li>加入50μLPBST覆盖的心脏样品，并培育在湿盒在5分钟的摇摆运动缓慢。重复冲洗更多的时间。这一步后，心脏样本，可以存储在湿盒1周在4 ° C </li><li>座与10％PBST羊血清在室温下1小时的心脏样本。 </li><li>与初级抗体，如β- catenin抗体，1:200稀释1:200稀释MEF2特异性抗体，孵育心脏样本，10％的羊血清/ PBST在室温下1小时。 </li><li>洗PBST三次在室温下（5分钟）的心脏样本。 </li><li>二次孵育样品1:1000稀释Alexa的，标签的反鼠标和/或抗兔抗体，如抗体，在PBST为0.5小时。 </li><li>用PBST洗三次心脏样本（5分钟）在室温下。 </li></ol> 4。成像<ol><li>取出PBST，每孔加10μL安装介质。摇滚室几分钟，直到解决方案变得清晰。 </li><li>取出安装介质，并涵盖所有的水井在一个用显微镜盖玻片（24x50）的幻灯片。 </li><li>图片鱼的心脏，使用蔡司Axioplan 2显微镜配备ApoTome（卡尔蔡司）。 </li></ol> 5。代表性的成果如图图像显示所有斑马鱼心肌细胞的细胞膜和核的一个例子。 1。 MEF2C和β-连环蛋白抗体染色52 HPF斑马鱼胚胎解剖心脏样本一起使用。虽然MEF2C抗体污渍，β- catenin的心肌细胞的细胞核抗体显示的每个细胞8-10边境。通过调整阶段的复式显微镜，图像的心脏样本，可以进行调整，以相同的方向，这将允许外曲率和曲率在心脏 11内观察。然后可以测量总的心肌细胞数量，心肌细胞大小和圆。 另一个例子，它揭示的一个胚胎的心脏横纹肌结构如图。 2。我们进行了免疫使用的F59，肌球蛋白抗体，胚胎心脏在解剖。在一个整体的胚胎心脏肌原纤维网络，可以清楚地表明在较低的放大倍率，而粗丝纹状波段可显示在更高的放大倍率（图2）6 。 <img alt="图1" src="/files/ftp_upload/3510/3510fig1.jpg" /> 图1。MEF2（红色）和β-连环蛋白（绿色）免疫染色显示细胞核和OUTLINES在斑马鱼心室的心肌细胞。比例尺=10μm的<img alt="图2" src="/files/ftp_upload/3510/3510fig2.jpg" /> 图2。肌球蛋白Immnostaining显示在斑马鱼心室粗丝或低倍率（A）或（二）高倍率比例尺=10μm的。

<ol>
	<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>

没有利益冲突的声明。

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 ...

immunostaining-of-dissected-zebrafish-embryonic-heart

Research

JoVE Journal

Biology

17.6K 次观看.  Mayo Clinic College of Medicine. 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。

视频: Immunostaining of Dissected Zebrafish Embryonic Heart

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

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 developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

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

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

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

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

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

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

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

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 fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

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

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

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

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

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

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

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

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

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

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

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

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

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

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

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 ...