Name: microdissection of zebrafish embryonic eye tissues
Uploaded: 2010-06-27T23:34:00
Description: Watch this Scientific Journal Video about Microdissection of Zebrafish Embryonic Eye Tissues at JoVE.com

这项工作是由一个在美国普渡大学生物科学系的启动资金支持。

第1部分：显微切割前的筹备工作<ol><li>解决方案<ol class="lalpha"><li> E3的中型4（5毫米氯化钠，0.17 mM的氯化钾， 氯化钙 0.33毫米和0.33毫米硫酸镁 ）。 </li><li>林格氏液1，5（116毫米氯化钠，氯化钾2.9毫米，1.8毫米氯化钙2，5毫米HEPES，PH7.2），过滤消毒。 </li><li> 5N氢氧化钠。 </li></ol></li><li>化学蚀刻钨针<ol class="lalpha"><li>安全小烧杯中，在培养皿中含有粘土5N?...

<ol>
	<li>Leung, Y. F., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+zebrafish+embryonic+retina.">Gene expression profiling of zebrafish embryonic retina.</a> Zebrafish. 2, 269-283 (2005).</li><li>Leung, Y. F., Ma, P., Link, B. A., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factorial+microarray+analysis+of+zebrafish+retinal+development.">Factorial microarray analysis of zebrafish retinal development.</a> Proc Natl Acad Sci U S A. 105, 12909-12914 (2008).</li><li>Leung, Y. F., Ma, P., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+zebrafish+embryonic+retinal+pigment+epithelium+in+vivo.">Gene expression profiling of zebrafish embryonic retinal pigment epithelium in vivo.</a> Invest Ophthalmol Vis Sci. 48, 881-890 (2007).</li><li>Nusslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish : a practical approach. , (2002).</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=The+zebrafish+book+:+a+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">The zebrafish book : a guide for the laboratory use of zebrafish (Danio rerio).</a> , (2000).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203, 253-310 (1995).</li><li>Fadool, J. M., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+model+system+for+the+study+of+eye+genetics.">Zebrafish: a model system for the study of eye genetics.</a> Prog Retin Eye Res. 27, 89-110 (2008).</li></ol>

斑马鱼眼组织显微切割可以有效地获得完整的视网膜和视网膜色素上皮附加视网膜。这大大助攻表达的研究，涉及到一个特定的眼组织（即视网膜色素上皮）。事实上，我们已经成功地利用这些程序获得整个视网膜和视网膜色素上皮 3 RNA的表达谱。这些配置文件的实用工具，是我们最近的途径和基因家族在视网膜分化突变2扰动识别，大力支持。在视网膜的解剖程序的最关键步骤?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cordless pestle motor</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>47747-370</td>
<td>&nbsp;</td>
<tr>
<td>DC power supply</td>
<td>&nbsp;</td>
<td>Lascar</td>
<td>PSU130</td>
<td>Any DC supply would work. The specific voltage of a different machine will need further optimization.</td>
</tr>
<tr>
<td>Disposable pestle &amp; microtube, 1.5 mL (DNase, RNase and pyrogen-free)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>47747-366</td>
<td>These are used for tissue collection in TRIzol for expression analysis.</td>
</tr>
<tr>
<td>Dumont #5 forceps, Tips: 0.05 x 0.01mm, Inox</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>500341</td>
<td>Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.</td>
</tr>
<tr>
<td>Dumont #5SF forceps, Tips: 0.025 x 0.005mm, Inox</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-00</td>
<td>Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.</td>
</tr>
<tr>
<td>Falcon polystyrene culture plates, 60 X 15 mm</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>351007</td>
<td>These plates are used as dissection plates.</td>
</tr>
<tr>
<td>Olympus SZX16 Stereomicroscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>SZX16</td>
<td>Any stereomicroscope would work. We used Leica stereomicroscope in previous studies1-3 without any issues. We also use the 1X objective exclusively for the dissection even though we have a 2X objective installed. </td>
</tr>
<tr>
<td>Sharpening stone</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>29008-01</td>
<td>Use this to sharpen the tip of the forceps if necessary</td>
</tr>
<tr>
<td>Thermo plate</td>
<td>&nbsp;</td>
<td>Tokai Hit</td>
<td>MATS-U55SZX2B</td>
<td>This is used to maintain the temperature of the tissue throughout dissection and minimize the influence of temperature fluctuation on gene expression. We also put the whole microscope in an environmentally controlled room at 28&deg;C during dissection in previous studies1-3 with good success.</td>
</tr>
<tr>
<td>Trizol, 100 mL</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15596-026</td>
<td>&nbsp;</td>
<tr>
<td>tungsten wire, 0.015 inch diameter</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TGW1510</td>
<td>&nbsp;</td>
<tr>
<td>Wooden Applicator</td>
<td>&nbsp;</td>
<td>Puritan</td>
<td>807</td>
<td>This is used for holding the chemically-etched tungsten needle.</td>
</tr>		</tbody>
		</table>
		</div>

microdissection of zebrafish embryonic eye tissues

斑马鱼眼睛发育的模式，因为其快速流行的动物进行研究<em&gt;前宫内</em&gt;发展和良好的繁殖力。受精后3天（DPF），幼虫会显示第一视觉反应。许多基因已被确定为控制适当的眼球发育，但我们正在从一个潜在的遗传结构的完整的理解远。全基因组基因表达分析是一个有用的工具，以澄清对眼睛发育的基因调控网络。然而，在斑马鱼的胚胎眼的小尺寸使得它具有挑战性的，以取得完整和纯粹的眼部组织表达分析。例如，2和第3天之间的眼睛前后的长度仅约为200-300微米，而镜头的直径为100微米少。此外，基本视网膜视网膜色素上皮（RPE）仅仅是一个单层上皮。虽然可以从整个胚胎中获得的基因的表达谱，他们不准确地代表这些组织中的表达。因此，纯粹的组织必须得到一个成功的眼睛发育的基因表达图谱。为了解决这个问题，我们已开发出一种方法来microdissect附1-3 DPF与视网膜色素上皮完整的视网膜和视网膜，其中包括眼的形态发生的主要阶段。所有的程序可以做到的标准立体显微镜下用细镊子和一般实验室用品。视网膜剥离，单层RPE细胞被删除，去皮刷牙行动和培养板剥离面的视网膜色素上皮残余的优惠坚持。对于视网膜色素上皮附加视网膜剥离，视网膜色素上皮坚持夹层板被删除之前，剥离，使RPE细胞可以与视网膜完全保留。一个小心解除这个组织的行动能够有效地分离推定脉络膜和巩膜。镜头可以在这两种情况下，由化学蚀刻钨针删除。总之，我们的方法可以获取完整的眼部组织，并已成功地利用研究斑马鱼视网膜组织特异性表达谱<sup&gt; 1，2</sup&gt;和视网膜色素上皮细胞<sup&gt; 3</sup&gt;。

Watch this Scientific Journal Video about Microdissection of Zebrafish Embryonic Eye Tissues at JoVE.com

Microdissection of Zebrafish Embryonic Eye Tissues

本文介绍的方法microdissect与视网膜色素上皮细胞附着，从一到三天受精胚胎斑马鱼视网膜。

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 ...

Research

JoVE Journal

Biology

Laser Capture Microdissection of Mammalian Tissue

激光捕获显微切割哺乳动物组织

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

科研

生物学

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 ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

在斑马鱼和镜头移植在眼突变分析中的应用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

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 ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Microdissection of Black Widow Spider Silk-producing Glands

显微切割的黑寡妇蜘蛛丝绸生产腺体

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

Immunostaining of Dissected Zebrafish Embryonic Heart

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 ...

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 ...