支持青少年糖尿病研究基金会授予5-2007-97（VEP），这项研究是由国家糖尿病，消化道和肾脏疾病研究所授予R01DK064973（VEP），R01DK48494（LHP），T32DK07074（支持标准煤），K01DK083552 （MDK），P60DK20595到芝加哥糖尿病研究和培训中心大学。内容完全是作者的责任，并不一定代表NIDDK或美国国立卫生研究院的官方意见。

1。注射前准备工作<ol><li>快速的鱼至少24小时前注射。这将空肠道灯泡（胃）的内容。基本空腹协议转让的鱼，在他们的正常密度，一个干净的坦克，然后扣的食物。对于较长期的空腹，需要更严格的条件下（如血糖研究），看到讨论的其他因素。 </li><li>准备盐溶液科特兰（Perry 等 ，1984）。 对于一个100毫升的量，溶于蒸馏水中以下内容： 725毫克盐（124.1毫米） 38毫克氯化钾（5.1毫米） 41毫克钠2 HPO 4（2.9毫米） 24毫克硫酸镁4∙7H 2 O（1.9毫米） 16毫克氯化钙2∙2H 2 O（1.4毫米） 100毫克碳酸氢钠3（11.9毫米） 4克聚乙烯吡咯烷酮（PVP）（4％） 千药典单位肝素 过滤，消毒，并保存在4 ° C。 </li><li>准备在显微镜。 <ul><li>盖保鲜膜在泄漏的情况下保护显微镜的基础。 </li><li>把保鲜膜的顶部纸巾。坐在纸巾上手术台。 </li><li>预调整的重点，通过观看手术表，并专注于海绵。 </li></ul>提示：把海绵上你的手指，并侧重于。这将进一步消除或减少焦距调整一次上手术台的鱼。 </li><li>称取鱼。 <ul><li>填写一个500 mL烧杯中约1 / 3充满鱼设施水。 </li><li>皮的平衡。 </li><li>使用网收集的鱼。威克多余的水远离简单地涂抹在纸巾上的净净鱼。鱼转移到烧杯中。 </li><li>称取鱼。 </li><li>鱼转移到一个干净的坦克。 </li><li>每个重达鱼转移到自己的标记坦克。 </li><li>计算的注射量为鱼体重的每条鱼。 </li></ul></li><li>准备的注射器和相关的注射设备。注射用，我们建议一个35G斜角钢针和10μLNanoFil microsyringe。准备NanoFil注射器制造商的指示和silflex油管。重要的是要删除任何气泡从注射器和管。灌装注射器和油管后，装入注射器泵上的，并计划第一条鱼的注射量。 </li><li>准备手术的表。 <ul><li>切柔软的海绵（如＃L800 - D Jaece工业），因此，它是在高度约20毫米。在平坦的脸，作出削减10-15毫米深。这种切割是注射的鱼槽，将举行。 </li><li>设置成60毫米培养皿的海绵。 </li><li>设置成一个适当大小的枪头箱盖海绵的培养皿。盖子必须大到足以容纳的水，以帮助保持海绵温度，但它应该是足够浅的方式获得。我们使用从P200的提示框是11.4厘米长x 7.7厘米宽x 1.5厘米的盖子D. </li></ul>这三个项目组装在一起（在培养皿中盘箱盖海绵）构成的手术台。 </li><li>准备麻醉剂。 <ul><li>请使用鱼设施水制成的多维数据集的碎冰。 </li></ul>提示：使用典型的冰块托盘，将采取3盘麻醉10-12鱼。 <ul><li>碎冰填充清洁冰桶。 </li><li>放入一个更大的容器，如一个2.4升Rubbermaid的食物储存容器的手术台。 </li><li>倒入一些设施水（温）外部容器和手术台。附近保持温暖设施，水的储备。 </li><li>外部容器放入一个温度计。 </li></ul></li></ol> 2。麻醉，注射和恢复<ol><li>将麻醉的外层容器加手术台旁显微镜。有冰块的桶附近。 </li><li>把水的温度下降到17℃加入冰块。重要提示：不要低于17 ° C这一步。 </li><li>使用净鱼转移到外部容器。 </li><li>慢慢加入冰块的容器，把温度降低到12℃，超过几分钟的过程中。 </li><li>监视器鱼的行为：在17 ° C或略低，鱼通常会传播其胸鳍水平，全球航空安全计划，并具有快速的鳃盖运动。随着气温下降，鱼会游泳更慢，最后停止游泳。由于手术的麻醉平面接触，喘气将停止，鳃盖运动将放缓。这种鱼是准备注射时没有反应处理。对于大多数鱼，12 ° C就足够了。更大的鱼可能需要更冷的WA之三。 </li><li>由于所需的温度达到饱和的海绵（〜12 ° C或更低），按。 </li><li>保持你的手指在冷水足以使他们不会热身鱼和带来的麻醉处理过程中。 </li><li>用冷的手指，轻轻的鱼转移到槽的海绵。腹部和槽鳃鱼的位置。 </li><li>快速传输的手术台，在显微镜阶段。 </li><li>工作迅速，小心地插入针腹鳍之间的中线。针指向颅插入肛门比接近骨盆带。你应该能感觉到，当针深体壁。注入适当的数量和撤出针。 </li><li>注射后，立即将鱼放回温水（〜28.5℃）回收罐释放出的鱼在鱼缸水的海绵。 提示：如果鱼没有立即开始游泳，帮助它恢复对鳃的水轻轻摇动。 </li><li>检查针。偶尔可连接规模和未来注射前应删除。 </li><li>对于随后的注射，用温水设施水，使麻醉室水温高达17℃前引入未来的鱼。 </li></ol> 3。代表性的成果： <img src="/files/ftp_upload/2126/2126fig1.jpg" alt="图1" /> 图1。代表性的结果，下面的腹腔注射0.5毫克/克葡萄糖或车辆。注射前72小时禁食鱼。 X轴显示时间，注射后。平均± SEM。

<ol>
	<li>Perry, S. F., Davie, P. S., Daxboeck, C., Ellis, A. G., Smith, D. G., Hoar, W. S., Randall, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+methods+for+the+study+of+gill+physiology.">Perfusion methods for the study of gill physiology.</a> Fish Physiology Volume X: Gills, Part B: Ion and Water. , 325-388 (1984).</li><li>Brown, L. A., Stoskopf, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+restraint.">Anesthesia and restraint.</a> Fish Medicine. , 79-90 (1993).</li><li>Eames, S. C., Philipson, L., Prince, V. E., Kinkel, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sugar+measurement+in+zebrafish+reveals+dynamics+of+glucose+homeostastis.">Blood sugar measurement in zebrafish reveals dynamics of glucose homeostastis.</a> Zebrafish. 7, 205-213 (2010).</li><li>Chavin, W., Young, 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=Factors+in+the+determination+of+normal+serum+glucose+levels+of+goldfish+Carassius+auratus+L.">Factors in the determination of normal serum glucose levels of goldfish Carassius auratus L.</a> Comp Biochem Physiol. 33, 629-653 (1970).</li><li>Groff, J. M., Zinkl, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematology+and+clinical+chemistry+of+cyprinid+fish.+Common+carp+and+goldfish.">Hematology and clinical chemistry of cyprinid fish. Common carp and goldfish.</a> Vet Clin North Am Exot Anim Pract. 2, 741-776 (1999).</li><li>Brand, M., Granato, M., Nusslein-Volhard, C., 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=Keeping+and+raising+zebrafish.">Keeping and raising zebrafish.</a> Zebrafish: A Practical Approach. , 7-37 (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=.">.</a> The Zebrafish Book. , (1995).</li><li>Iwama, G. K., Ackerman, P. A., Hochachka, P. W., Mommsen, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetics.">Anaesthetics.</a> Biochemistry and Molecular Biology of Fishes, Volume 3: Analytical Techniques. , 1-15 (1994).</li><li>Reavill, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+diagnostic+and+clinical+techniques+for+fish.">Common diagnostic and clinical techniques for fish.</a> Vet Clin North Am Exot Anim Pract. 9, 223-235 (2006).</li><li>Stoskopf, M. K., Stoskopf, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgery.">Surgery.</a> Fish Medicine. , 91-97 (1993).</li></ol>

腹腔注射包括五个步骤：禁食，称重，麻醉，注射，和恢复。对于每个步骤，有最好的做法，可以确保成功。成功包括健康的鱼的病人，以及良好的实验结果。 空腹：24小时快速清空肠道灯泡。这种做法是采取从鱼兽医文献（例如，1993年布朗）。附加空腹考虑下面讨论。 较长期禁食：我们已经找到，需要72小时快速注射前降低血糖至基线水平（埃姆斯等 ，2010）。我们还发现，对葡萄糖的研究中，有几个程序，所需，以确保鱼停食正确。用干净的坦克（没有在底部的碎片）开始。坦克应该是离线的，清楚地标示为“空腹”，并在一个位置，热情的鱼医护人员将不能养活他们。评估坦克的外部环境，并采取措施防止鱼从干扰强调，强调的是已知的，以提高血糖（Chavin和杨，1970;。Groff的等 ，1999 ）。例如，我们有一个空腹的实验中，一台收音机，每天工作在板凳上，拿着鱼缸。我们发现，血糖异常高，并得出结论，鱼振动强调。另一种应激是人满为患。鱼应保持在好鱼的饲养方法，符合密度。有关建议，请参阅品牌等 。 （2002年）和韦斯特（1995年）。我们有良好的效果，空腹我们在9升的油箱（3层占用一些体积大理石）的密度在10-12鱼的鱼。两性分离，可能会造成压力，所以我们建议维持一个混合性的人口在快速。这意味着可以奠定鸡蛋，鸡蛋需要被隔离，使他们不会被吃掉。封存鸡蛋一个简单的方法来覆盖2-3层大理石罐底。需要通过去除鸡蛋和废物，每天更换水箱的水大约10-15％，保持水质。对于去除鸡蛋和浪费，虹吸效果很好。 称重：称重鱼类，不麻醉时，应小心放入烧杯中，以尽量减少净调水，以保证称量准确。如果纸巾吸干网（鱼），大部分多余的水可以被删除，并可以准确测量体重。称重前麻醉鱼可能会更容易，但我们没有一天两次鱼的麻醉可能造成的影响进行测试。我们有我们的技术测试，称重网/印迹方法，然后重新称重的鱼的鱼后，已麻醉，轻轻涂抹干。我们没有发现的方法之间的重量显着性差异（P = 0.7927，t检验）。此外，我们测试是否扣除/印迹方法比较简单的鱼转移到烧杯中，只要它是落网（无杂交）受影响的血糖。我们没有发现两者之间的传输方法在血糖水平的显着性差异（P = 0.2241，t检验）。 Anesthetizing：化学麻醉可能适用于许多研究。在这里，我们已经证明作为替代冷水麻醉，因为许多麻醉药（包括tricaine/MS-222（布朗，1993年）），提高血糖。在以前的研究中，我们有决心，冷水不提高血糖，在斑马鱼（ 埃姆斯等，2010） 。 冷水麻醉，温度应缓慢下降。减少的幅度似乎取决于鱼的大小，较小的鱼下速度比更大的鱼。注射后，您可能会发现，鱼是从麻醉中恢复速度太慢（见下文）。这可能会导致当起始温度太低，或当温度下降过快。起始温度过低，入水后，如果鱼的横向弯曲。如果起始温度是正确的，鱼最初将保持其平衡。它会旋转到水平位置的胸鳍，喘气，并具有快速的鳃盖运动。通常情况下，它会游泳。随着温度的降低，动作减少，鱼会失去平衡。手术的麻醉平面到达时，鱼可以处理没有反应。为了维持手术麻醉下的鱼，你的手指必须是冷的，所以他们在水中前处理鱼。海绵也必须保持在同一温度下用于麻醉的鱼水作为冷。重要的是要饱和与水的海绵，是sufficiently冷维持麻醉，一旦鱼放置到它。 注射液：进行注射前，您可能要解剖至少有一条鱼，体壁的厚度感。这可以帮助你判断多远针需要插入进入​​腹腔。此外，当您插入针，你可以感受到身体墙“给”，当针进入腹腔。注射期间，采取措施，使病人高兴。确保饱和的海绵是正确的温度冷水，以防止注射过程中复苏的鱼。尽量减少破坏的规模和粘液覆盖的皮肤是很重要的一个饱和和柔软的海绵。同样重要的是保持鳃通气良好的饱和的海绵。我们强烈建议下面列出根据材料的海绵。最后，一​​旦鱼被麻醉，迅速开展工作，以尽量减少鱼是下的时间。 恢复：鱼应该从麻醉中恢复过来，几乎进入了温暖的鱼缸水。如果鱼没有开始立即游泳，轻轻对鳃，加快恢复的漩涡水。如果复苏缓慢，然后鱼去下得太快，你应该适当调整麻醉过程。下麻醉缓慢复苏的可能原因进行了讨论。

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Foam Sponge</td>
<td>&nbsp;</td>
<td>Jaece Industries</td>
<td>L800-D</td>
<td>&nbsp;</td>
<tr>
<td>60 mm Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipet tip box lid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>not too deep, e.g. 1.5 cm</td>
</tr>
<tr>
<td>Plastic storage container</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>deep, e.g. 7 cm </td>
</tr>
<tr>
<td>Thermometer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Crushed ice</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>made from facility water</td>
</tr>
<tr>
<td>Warm facility water</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1 liter or more</td>
</tr>
<tr>
<td>500 ml beaker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>for weighing</td>
</tr>
<tr>
<td>NanoFil syringe</td>
<td>&nbsp;</td>
<td>World Precision Instruments (WPI)</td>
<td>NANOFIL</td>
<td>or Hamilton syringe</td>
</tr>
<tr>
<td>35 gauge needle</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>NF35BV-2</td>
<td>beveled</td>
</tr>
<tr>
<td>Silflex tubing</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>SILFLEX-2</td>
<td>&nbsp;</td>
<tr>
<td>UltraMicroPump III and Micro4 controller</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>UMPS-1</td>
<td>&nbsp;</td>
<tr>
<td>Foot switch</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>15867</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plastic wrap</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Paper towels</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cortland salt solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

intraperitoneal injection into adult zebrafish

化学治疗斑马鱼的一个方便的方法是引入水箱水，它会采取鱼试剂。但是，这种方法使人们难以知道的试剂吸收或每鱼。一些实验性的问题，特别是那些与代谢研究，可能会得到更好的解决提供了一个定义每条鱼的数量，重量。在这里，我们提出了一个腹腔（IP）注射到成年斑马鱼的方法。注射到腹腔，骨盆带后。此过程是改编自兽医的方法，用于更大的鱼。它是安全的，正如我们所观察到的零死亡率。此外，我们已经看到在只有5 127注射的注射部位出血，并在这些情况下，每年的出血是短暂的，持续几秒钟的时间，失血量小。这个过程的成功，需要通过几个步骤，包括禁食鱼温柔处理，称重，anesthetizing，注射和恢复。需要注意事项，以尽量减少在整个过程中的应力。我们的预防措施，包括使用一个小的注射量和35G针。我们使用的车辆，这是渗透平衡淡水鱼科特兰盐溶液。首先把手术的麻醉平面，它允许缓慢的鳃盖运动的鱼鳃曝气保持在注射过程，和第二，通过控股处于低谷的鱼在水饱和的海绵，在注射本身。我们通过注射葡萄糖和监测在血糖升高，随后恢复正常的IP注射的效用。由于压力是已知会增加血糖骨鱼，我们比较车辆注射和非注射的成年人血糖水平，并显示该程序不会导致血糖显着上升。

Watch this Scientific Journal Video about Intraperitoneal Injection into Adult Zebrafish at JoVE.com

Intraperitoneal Injection into Adult Zebrafish

我们证明到成年斑马鱼的腹腔注射。我们使用10μLNanoFil microsyringe由一个Micro4控制器和UltraMicroPump第三控制。此演示包括作为麻醉剂使用的冷水。

腹腔注射到成年斑马鱼

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

intraperitoneal-injection-into-adult-zebrafish

Research

JoVE Journal

Biology

57.0K 次观看.  The University of Chicago. 我们证明到成年斑马鱼的腹腔注射。我们使用10μLNanoFil microsyringe由一个Micro4控制器和UltraMicroPump第三控制。此演示包括作为麻醉剂使用的冷水。

视频: Intraperitoneal Injection into Adult Zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

在成年斑马鱼的全肾骨髓移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

科研

生物学

Injection of dsRNA into Female A. aegypti Mosquitos

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus.  The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated.

女答：埃及伊蚊蚊的dsRNA注入

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

斑马鱼脑室注射液

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

在成年斑马鱼的眼窝射出

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

剖析器官的成年斑马鱼

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

通过注射和电穿孔的斑马鱼Otocyst的MIF Morpholinos直接运抵影响内耳发展

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

直接进入成年斑马鱼的肾细胞移植

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

成年斑马鱼肾脏的解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

再生的成年斑马鱼尾鳍在吗啉代入体内电穿孔

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...