The authors have nothing to disclose.

所有的程序都按照“实验动物福利原则”所述的准则进行（卫生出版没有。85-23国家研究院​​，1985年修订）和经批准的罗莎琳德富兰克林大学实验动物管理和使用委员会动物协议08-19。 1，庆大霉素诱导的毛细胞坏死<ol><li>制备硫酸庆大霉素生理盐水在0.004％的终浓度（4.32毫摩尔）。 </li><li>在含有0.004％（4.32毫米）庆大霉素溶液的容器放置成鱼（ 斑马鱼 ，4-6个月的婴儿）。任何容器均可使用，但我们使用鱼容器从医药品水产系统是在宽7所示，在高6，和在长7。将容器鱼在孵化器设定在28℃，24小时。在足够的电平设置流体在罐的总容积在一个可行的状态保持鱼的24小时期间。注：庆大霉素液的曝气不是NECEssary如果有足够的容积用于鱼被处理的数量。 </li></ol>毛细胞的2。活体染色<ol><li>制备的荧光活体染料[4 - 4 - 二乙基氨基）-N-甲基吡啶鎓碘化物（485nm激发λ和甲醇603 nm发射λ）从15毫克/毫升的工作原液的0.08％的浓度（在生理盐水中）乙醇。 </li><li>以确定是否庆大霉素治疗是有效的控制和庆大霉素的一个子集处理的鱼是由在含有活体染料6孔培养板的孔放置鱼立即染色。根据需要为要达到统计学显着性使用鱼的足够数量（培养板，基于神经丘计数检查者的速度，将鱼在所述板以交错的方式随着时间的推移，这样的鱼不染色，在75分钟按照步骤2.3中所述。 </li><li>通过荧光显微镜放置板从一个抽屉里板凳2.2步可用于检查神经丘染色的。关掉房间的灯，以防止活体染料的猝灭过在室温下1小时，染色时间。 </li><li>准备两种染料洗出和麻醉水箱。染料洗出的水是正常的鱼和水的麻醉水，补充足2 - 苯氧基乙醇，这样一1:1000稀释在正常的鱼水的实现。 </li><li>将鱼超出正常的鱼水冲洗多余的活体染料，并继续执行步骤3.1观察生命染料染色的鱼。 </li><li>为了研究神经丘的再生，转移在28°C洗涤正常的鱼水的孵化8-16小时之间的庆大霉素处理的鱼</li><li>在8-16小时之间的不同时间，鱼从培养箱中取出，洗涤，染色按照步骤2.1-2.4所示。继续执行步骤3.1观察生命染料染色的鱼。 </li></ol> 3，麻醉测定鱼和神经丘的荧光计数<ol><li&gt;吸干纸巾上每条鱼去除多余的液体，然后将其放置在一个湿润的滤纸是集中在塑料培养皿的盖子。 </li><li>放置在荧光立体显微镜阶段的盖子，得到的中间体线圈的活体染料染色的神经丘的数字图像。 </li><li>使用数码相机放置在荧光立体显微镜载2X的放大率捕获图像用于后续的定量分析。注：立体显微镜的放大倍数设定可能取决于显微镜使用的品牌，但该设置应该让身体中缝内便于查看和个别神经丘的计数。 </li><li>通过计数在鱼刚好接近右胸鳍的最底层的腹侧四个指定的线圈内的可见神经丘的数量（参见图1）确定再生的量。统计分析使用适当的测试，如方差分析Ør中的学生T检验。实验应利用最少的每个时间点5鱼和所有的实验应重复至少3倍的。 </li><li>基于神经丘再生时间曲线（ 见图3），数8-16小时庆大霉素后洗出的神经丘是再生曲线的线性相位内。注意：使用线性时间阶段允许控制组和实验组之间的正确定量分析。 </li></ol>个别毛细胞4。为获得更高的分辨率定量分析的，如果神经丘分析无统计学意义荧光计数<ol><li>如果在神经丘水平的定量分析是不显著，分析在单个毛细胞的电平也可以被用来获得较高程度的分辨率。在特定的时间点庆大霉素后洗出（时间点的基础上，早期的神经丘研究），活体染料ST选择鱼泉如在方案2中所述的鱼，然后用2 - 苯氧基乙醇以1:500稀释为1-5分钟安乐死的鱼。 </li><li>在柔和的光，以防止淬火，使4切口，使得正方形皮瓣制备是如下进行的。做一个切口沿鱼的上肋，直到它与肛门鳍对准，然后使整个腹部切口，最后，这样的方皮瓣被创建使这些切口的每一侧上的两个垂直切口。注意：这种皮肤准备工作将纳入在神经丘实验中使用的身体中针。 </li><li>将皮肤标本在载玻片上，然后将一个圆形玻璃盖片在皮肤切除标本帮锚杆和扁平化的组织进行后续的数字成像。 </li><li>利用皮肤标本从步骤4.3，获得毛细胞的中间体的线圈的每个神经丘内的数字图像。拍摄图像在至少60X的放大倍数再算上头发CE单个神经丘内LLS为对照组和实验组的比较定量分析（ 见图4）。 </li></ol>

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This work was supported by a research grant from the Iacocca Family Foundation, National Institutes of Health Grant DK092721 (to R.V.I.), and Rosalind Franklin University start-up funds. No potential conflicts of interest relevant to this article were reported. G.C.P., S.M.M, and N.D. researched data. M.P.S. Jr. and RI oversaw the project, contributed to the discussion of the data, and oversaw the writing and editing of the manuscript.

基于已建立的侧线（LL）再生的胚胎和幼虫斑马鱼8,24,25分析文献的广泛身体，我们的研究目标是开发在斑马鱼的定量检测横向线条的再生可能被应用到被研究最好在成年鱼的疾病模型。我们发现，应用到成鱼开发的胚胎/仔鱼过程时，某些关键点是很重要的。最重要的这些点被认为：1）侧线的数量沿鱼的纵向轴线神经丘，神经丘毛细胞染色的2），3）治疗的氨基糖苷的浓度和持续时间，和4...

侧线（LL）系统是鱼类和两栖类，它负责听觉，平衡，rheotaxis调处行为，如教育和捕食者回避1-5找到了mechanosensory器官。这两者它是由簇毛细胞，支持细胞包围，被定位在结构，称为神经丘6。这些神经丘通常组织成垂直线（称为针）沿主体和尾部与一些在鱼的头部观察到的水平线圈的纵向轴线。在成人中，神经丘是显著更大数目的线圈相比，胚胎或仔鱼6内。在斑马鱼生物医学研究都集中在抗生素治疗，噪音引起的创伤，慢性感染等的影响。对毛细胞，试图7,8，以便更好地了解他们在人体的影响。 与大多数脊椎动物，德硬骨鱼，如斑马鱼（ 斑马鱼 ），必须重新生成丢失的毛细胞的能力。因为它们的快速开发时间和高再生能力的斑马鱼是特别有用的。迄今为止，然而;侧线上的开发和/或再生斑马鱼研究主要利用的胚胎和幼虫阶段的鱼，由于侧线神经丘的数量减少可以较容易计数和分析6,9,10。 然而，神经系统和非神经系统疾病11-16尽可能多的斑马鱼模型研究了成鱼，而不是幼虫，我们专注于开发在成年斑马鱼用庆大霉素横向线再生法（以前在斑马鱼幼虫用一种氨基糖苷类和最近与成年鱼17用），这样的测定法是可用的，可以被应用到当前的成年斑马鱼的疾病模型。而此前公布的程序由Van特朗普ét铝，17设立在成鱼的毛细胞消融的条件，他们没有建立标准曲线这是需要控制组和实验组利用转基因斑马鱼线或药物诱发的疾病状态时，如之间的定量比较神经丘再生在斑马鱼18。因此，我们跟着范·特朗普等人 17的毛细胞消融的程序，但建立在他们的工作，以建立神经丘再生的标准曲线，以使研究者比较对照组和实验组在使用我们的数据，如与成年斑马鱼疾病模型。该测定也被设计为允许扩展的分析对个体毛发细胞时，需要解决的一个更高的水平。

<table border="0" cellpadding="0" cellspacing="0" width="431">
	<tbody>
		<tr>
			<td>Gentamicin sulfate solution (50mg/ml)</td>
			<td>Sigma Aldrich</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Phenoxyethanol</td>
			<td>Sigma Aldrich</td>
			<td>P1126</td>
			<td></td>
		</tr>
		<tr>
			<td>4-4-diethylaminostryryl-N-methylpyridinium iodide (4-Di-2-Asp)</td>
			<td>Aldrich</td>
			<td>D-3418</td>
			<td>485 nm excitation &#955; and 603 nm emission &#955;</td>
		</tr>
		<tr>
			<td>in methanol</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Mid Sci</td>
			<td>TP92006</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>Matek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting&#160; Microscope</td>
			<td>Nikon</td>
			<td>TMZ-1500</td>
			<td>Any dissecting microscope is fine.</td>
		</tr>
		<tr>
			<td>Camera for Imaging</td>
			<td>Nikon</td>
			<td>Q imaging</td>
			<td>Any camera is suitable.</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td>NIH Image</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Any imaging software is suitable.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV10i</td>
			<td>Any high resolution fluorescent microscope is suitable</td>
		</tr>
		<tr>
			<td>Aquatic System</td>
			<td>&#160;KG Aquatics ZFS Rack System.</td>
			<td></td>
			<td>Any aquatic system can be used</td>
		</tr>
	</tbody>
</table>

an assay for lateral line regeneration in adult zebrafish

由于听力和平衡紊乱在人的临床重要性，模式生物如斑马鱼已被用于研究横向线的开发和再生。斑马鱼是由于其快速的开发时间和它的高再生能力为这些研究特别有吸引力的。迄今为止，侧线再生斑马鱼的研究主要是利用，因为在这几个阶段的较小的数字神经丘的胚胎和幼虫的鱼。这使侧线再生/和或更早发育阶段的开发更容易进行定量分析。由于神经系统和非神经系统疾病的许多斑马鱼模型研究了成鱼，而不是在胚胎/幼虫，我们专注于开发一个定量的侧线试验再生的成年斑马鱼，这样的分析是可用的，可以被应用到当前成年斑马鱼疾病模型。由Van TRUM建立在前人研究所描述的毛细胞在成年墨西哥洞穴盲鱼和斑马鱼（ 斑马鱼 ）的消融过程P 等17中 ，我们分析的目的是让控制组和实验组之间的定量比较。这是通过开发一种基于神经丘再现过以下毛细胞在限定的横向线的区域庆大霉素引起的坏死一个24小时时间段的百分比的再生神经丘标准曲线来实现。该测定也被设计为允许扩展的分析，以当需要的分辨率更高水平的个体毛发细胞的水平。

优化的程序，在量化成年斑马鱼的侧线神经丘再生。 斑马鱼幼虫的神经丘是容易量化;然而，成年斑马鱼的侧线具有更大数目的每针脚神经丘作出定量分析更加困难6,17,19,20的。就像在图1A中 ，头部有许多神经丘相比，任一中间部分或尾部的显著更高;用具有如图所示的一维数最少的神经丘的尾部区域。因为线圈中的头...

Watch this Scientific Journal Video about An Assay for Lateral Line Regeneration in Adult Zebrafish at JoVE.com

An Assay for Lateral Line Regeneration in Adult Zebrafish

由于神经和非神经系统疾病的许多斑马鱼模型研究在成年鱼，而不是胚胎/幼虫，我们开发了可应用于成年斑马鱼疾病模型的定量侧线再生试验。该法涉及的分辨率在1）神经丘和2）单个毛细胞的水平。

对于侧线再生成年斑马鱼的含量

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

an-assay-for-lateral-line-regeneration-in-adult-zebrafish

Research

JoVE Journal

Biology

12.5K 次观看.  Dr. William M Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science. 由于神经和非神经系统疾病的许多斑马鱼模型研究在成年鱼，而不是胚胎/幼虫，我们开发了可应用于成年斑马鱼疾病模型的定量侧线再生试验。该法涉及的分辨率在1）神经丘和2）单个毛细胞的水平。 

视频: An Assay for Lateral Line Regeneration in 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

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm.

The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.

Nanogenomedicine在MCF - 7细胞的细胞毒性：MTT法

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based ...

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 ...

Intraperitoneal Injection into Adult Zebrafish

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, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

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, ...

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 ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

在成年斑马鱼的生化分析采血车

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging成年斑马鱼

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

重复采血车在成年斑马鱼验血

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

在 Vivo 表面心电图中成人斑马鱼

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...