Name: live imaging of the zebrafish embryonic brain by confocal microscopy
Uploaded: 2009-04-01T00:00:00
Description: Watch this Scientific Journal Video about Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy at JoVE.com

<p class="jove_content">谁最先开发这项技术在Sive实验室的博士詹妮弗Gutzman许多感谢。达纳 - 法伯癌症研究所的波士​​顿，马萨诸塞，慷慨地提供CAAX - GFP基因的质粒编码也感谢JB绿色。白石WM凯克基金会生物成像设施进行共焦成像。 HS是由NIHMH70926支持。 EG是支持由美国国家科学基金会博士后奖学金。 </p>

<p class="jove_title"> 1。准备注射基因</p><ol><li>在此过程中使用的mRNA是由质粒编码CAAX - EGFP（memGFP）mRNA的转录。首先线性化的质粒，根据2008年等，Gutzman。 </li><li>然后抄写memGFP mRNA的使用mMessage mMachine套件。 </li><li>稀1μg/μl，导致mRNA的等份，并储存在-80 ° C。 </li><li>在一个细胞阶段的胚胎车道​​的宽度，准备1％琼脂糖的注塑模具。 </li><li>在注射前一天，成立了分离的女性，男性的交配笼。 </li><li>注射日，拉毛细管?...

<ol>
	<li>Gutzman, J. H., Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+zebrafish+midbrain-hindbrain+boundary+constriction+requires+laminin-dependent+basal+constriction.">Formation of the zebrafish midbrain-hindbrain boundary constriction requires laminin-dependent basal constriction.</a> <em style='font-style: italic'>Mech Dev</em>. <b>125</b> (974), 11-12 (2008).</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> <em style='font-style: italic'>The Zebrafish Book: a Guide for the Laboratory Use of Zebrafish</em>. , (1995).</li></ol>

<p class="jove_content">在这段视频中，我们表现出的mRNA注射到单细胞的斑马鱼胚胎的方法。在这里，我们使用一种膜定位GFP标记每个细胞的基因编码。然后，我们演示了如何安装和形象在单细胞分辨率的发育中的大脑。这项技术使我们能够研究一种新型的细胞形态变化，基部收缩，形成中脑，后脑边界（Gutzman等，2008）所需。类似的分析等现象有可能显着扩大我们对脊椎动物的形态发生和底层细胞生物学的理解。 </p>

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<td>SeaKem GTG agarose</td>
<td>   Reagent</td>
<td>Lonza</td>
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<td>&nbsp;</td>
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<td>3-aminobenzoic acid ethyl ester (Tricaine)</td>
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<td>Silicone vacuum grease</td>
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<td>Zeiss LSM 510 scanning confocal </td>
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<td>Micropipette puller</td>
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<td>Sutter Instruments</td>
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live imaging of the zebrafish embryonic brain by confocal microscopy

<p class="jove_content">在这段视频中，我们展示了我们的实验室已发展到分析细胞形态的变化和需要弯曲和折叠的发展斑马鱼脑（Gutzman等，2008）的重排的方法。这种分析提供了一次脊椎动物大脑的三维结构的发展所需的基本细胞生物学的新认识，并显着提高我们的能力，以研究神经管的形态发生。胚胎斑马鱼的大脑形脑室神经上皮内膨胀的开端，在18小时后受精（HPF）。 24 HPF，神经管的形态发生的最初步骤，是完整的。使用这里介绍的方法，在细胞阶段的胚胎基因编码的膜，有针对性的绿色荧光蛋白（memGFP）注射。后注射和孵化，胚胎，目前18至24 HPF，安装，倒置，在琼脂糖和激光共聚焦显微镜成像。值得注意的是，斑马鱼的胚胎是透明的，使其成为理想的荧光成像系统。虽然我们的分析集中在中脑，后脑边界和后脑，这种方法可以延长任何地区的分析，在深度为80-100微米的斑马鱼。</p>

Watch this Scientific Journal Video about Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy at JoVE.com

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

<p class="jove_content">在这段视频中，我们表现出的方​​法，通过它来分析生活在单细胞共聚焦显微镜分辨率的斑马鱼胚胎发育中的脊椎动物大脑。这包括我们注入单细胞的斑马鱼胚胎的方法，通过它，随后装载和图像的大脑发育。</p>

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 ...

Research

JoVE Journal

Biology

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

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科研

生物学

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Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

基于荧光测定活细胞中的钙进入商店经营：从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

成像和胚胎斑马鱼脑血管结构的三维重建

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

<em>In Vivo</em> 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

<em>在</em>造血干细胞和祖细胞的成年小鼠颅骨骨髓<em>体内</em>四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

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Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

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

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

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

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

侵入斑马鱼大脑的胶质母细胞瘤细胞微管动力学的实时成像

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...

Autofluorescence Imaging to Evaluate Red Algae Physiology

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Red algae (Rhodophyta) contain phycobiliproteins and colonize habitats with dim light, however some (e.g., some Chroothece species) can also develop in ...