Name: analysis of zebrafish larvae skeletal muscle integrity with evans blue dye
Uploaded: 2015-11-30T15:00:00
Duration: 7 min 34 s
Description: Watch this Scientific Journal Video about Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye at JoVE.com

我们要感谢特伦特沃他的技术援助。我们也承认儿科系病童医院和治疗先天性肌营养不良（CMD）的慷慨资助这一项目。

1.准备琼脂注射板（时间：45分钟） <ol><li>煮2％至3％琼脂糖在E3媒体和允许的解决方案，以在板凳上略有降温。注意：正在准备注射板的数量表明琼脂糖的需要量。每次注射板需要约35毫升琼脂糖溶液中。 </li><li>煮沸后，使琼脂糖冷却直到达到所需的温度（例如，45℃），为每注射模具制造商的说明。 </li><li>倒入约35毫升冷却的琼脂糖为100毫米的菜。 </li><li>将首选注塑模具到溶液中的一端，然后躺在模具的其余部分到琼脂糖溶液（这将有助于减少气泡的产生）。 </li><li>允许琼脂糖溶液固化或者在RT或4℃大约30分钟。 </li><li>用刮刀到模具的一个端部从所述固体琼脂糖分离。慢慢地取出m个剩余<html>旧。 </li></ol> 2.准备埃文斯蓝色染料（EBD）注射液混合的（时间：30分钟） <ol><li>使EBD 1％的库存在1X林格氏液（155毫摩尔NaCl; 5mM的氯化钾; 2毫米氯化钙2; 1mM的MgCl 2的; 2毫的 Na 2 HPO 4; 10mM的HEPES; 10mM葡萄糖; pH至7.2），它可以储存在室温。 </li><li>使异硫氰酸荧光素储备溶液（FITC） -葡聚糖分子量10000 kDa的25毫克/毫升在1X林格氏溶液并储存在-20℃。 </li><li>准备注射液混合在了FITC葡聚糖股票原液稀释EBD直接0.1％（即，对于最终工作100微升量：将10微升1％EBD的90微升FITC葡聚糖股）。 </li><li>彻底涡流喷射混合（它应该变为绿色），远离直射光通过包装注入混合管铝箔。 </li></ol> 3. EBD注射剂（时间：约30分钟） ENT“&gt;注：协议在最佳状态下3-7天受精后（DPF）的幼虫。 <ol><li>前期预热注射板RT。 </li><li>设置注射装置通过布置显微上的金属板和站旁的显微镜被用于注射。打开空气驱动显微注射控制器。注：优选的注射系统将通过实验室变化，并且不应该改变的分析结果。 </li><li>回填土注射针，大约2-4微升EBD组合。 </li><li>校准注射量为约5 NL EBD组合。注意事项：注射量校正将取决于校准方法。活塞驱动的注射可以直接设置到给定的注射量而气体压力喷射器将需要的注射体积通过量丸药与使用千分尺校准。 </li><li>湿式喷射板1X林格氏液，并从井中取出多余的。 </li><li>预治疗幼虫用0.04％的3-氨基苯甲酸乙酯甲磺酸盐萨尔吨（三卡因）稀释在1X林格氏液来固定在注射前的起始幼虫。注：确保幼虫完全immotile是重要的，因为正确的注射液是很难与任何残留的运动。 </li><li>放置麻醉幼虫，并将使用的玻璃吸移管的琼脂喷射板的孔。确保幼虫完全井内，躺在自己的身边。注意：每孔幼虫的数目是高达实验者。 </li><li>被放入孔后幼虫，除去过量的林格氏液在井内最小化幼虫运动。留下的溶液的残留量，以使得幼虫不脱水。 </li></ol>斑马鱼幼虫4.心包注射EBD（时间：取决于幼虫注射的数量，估计1-3个小时） <ol><li>上放置装有其中注射将执行解剖范围幼虫注入板。 </li><li>定位包含注射针针该EBD混合了斑马鱼幼虫。 </li><li>重新定位的注入板通过旋转它，以便将注射针靠近幼虫的心脏和大约45°腹侧从前后轴。 </li><li>将注射针头插入公共主静脉（CCV）在静脉的区域的蛋黄，其中静脉最初转动在背方向（图1）的前部。注：最高40倍的放大倍率可能有助于清楚地看到CCV。 </li><li>注入5 NL EBD组合，并保持注射针在位置5-8秒，以尽量减少直接泄漏EBD组合。注意：一个好的注射将有染料着色见于心脏腔室（ 图1）。如果EBD混合物中未观察到心脏，然后注入额外的5 NL EBD混合物可以是足以诱导染率。可替代地，胚胎可以被丢弃。 注：在某些情况下，心脏会停止跳动。如果这Øccurs，继续监视幼虫20-40秒。通常情况下，心脏跳动恢复作为染料通过循环系统移动。 </li><li>进入下一个幼虫和重复。 </li><li>通过注射（图2）后，立即观察的FITC-葡聚糖在脉管的存在识别成功注射的胚胎。 </li></ol> 5.孵化和EBD吸收（时间：4-6小时） <ol><li>后幼虫的期望数量的注入，返回在100mm平皿注入幼虫1X林格氏液无三卡因。 </li><li>保持菜肴用铝箔包裹。注意：保持注射幼虫在黑暗中显著提高生存率，并确保在信号强度最大的一致性。包裹在铝箔为的时间段，该幼虫的保育箱的外面尤其重要。 </li><li>允许幼虫孵化在28.5℃，4-6小时，以保证有足够的EBD摄取。 </li></OL&gt; 6，可视化EBD的肌肉（时间：取决于幼虫注入和显微镜的类型的数量，预计0.5-3小时） <ol><li>在成像前，麻醉幼虫用0.04％三卡因以防止移动。 </li><li>下红色荧光视图幼虫以确定是否EBD摄取是发生在骨骼肌（图3）。 </li></ol>

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作者有没有竞争经济利益或其他的利益冲突。

斑马鱼正在成为一个强大的工具，用于神经肌肉疾病2,29研究。迄今为止，斑马鱼系统已用于验证新的肌肉致病突变16,17,30，阐明新颖病理机制18，并识别潜在的新的治疗药物12,24。这些集体努力已经建立了斑马鱼的实用程序来模拟人类神经肌肉疾病。然而，尽管与斑马鱼和哺乳动物模型所取得的进展，有用于神经肌肉条件宽光谱内患者有限的治疗选择。因此，一个高需求存在治疗的发展，这组毁灭性的疾病。并联的治疗这种需求对应的需要不断实验创新，以及严谨的分析，以验证新的动物模型和假定的治疗策略。 EBD分析是常用的小鼠模型来研究组织和在脑，心脏细胞损伤，和骨骼肌27,31。最值得注意的是，EBD被广泛应用于各种肌营养不良症亚型的小鼠模型显示的肌膜不稳定的严重性和损坏8。使用EBD的揭示肌肉膜损伤是一个有利的参数建立动物模型的相似性对人类疾病状态9。 EBD在鼠标的电源已导致几个实验室，包括我们自己，开发和应用EBD神经肌肉疾病的斑马鱼模型。由于EBD分析的适用性，这种技术正在积极实施以证实斑马鱼的模型与人类疾病状态11,15,22,24,32。幼虫受损的肌肉的膜将具有肌纤维内EBD摄取，因此红色荧光。荧光在纤维间空间观察到的，但不是个别的肌肉纤维中也可以是信息性的纤维，从在吨基底膜分离的他没有膜的损伤，提供有用的诊断细节。 EBD分析已经超越动物模型验证的应用前景。从我们的实验室努力最近证明，EBD分析，有利于在确认潜在的新型治疗药物24。如果确定潜在的治疗治疗减少或取消EBD摄取神经肌肉疾病模型可以表示相关的治疗作用8。这种类型的分析可以帮助确定治疗的机制（S）和扩展EBD分析中的应用。 与许多技术，EBD分析确实有几个注意事项在实验设计和实践中加以观察。例如，它可以是具有挑战性的识别CCV由于随着年龄的组织的增厚。此外，它很容易损坏幼虫在制备之前和心包注射期间，减少实验计数和增加的需要来准备大量的幼虫。此外，处理和注射过程中所做的幼虫物理损坏可能会导致误报受损的肌肉会占用EBD。为了克服这些障碍，我们这个视频文章，它允许容易和可靠识别幼虫与成功的染料输注后立即注入和之前随后的分析中所描述的共注入策略。的FITC-葡聚糖共同注射对照成功注射通过允许在脉管确认EBD的之前，其吸收的肌肉纤维。作为EBD荧光变得在幼虫高度扩散，如果在肌肉纤维未收集几个小时后这可以是特别有用的;因此，它可能难以察觉。此外，缺少CCV和注入EBD入蛋黄或体腔可以培养后，导致弥漫性红色荧光类似于控制胚胎，但有吸收的受损的肌肉纤维的可能性降低。总的来说，这些洞穴ATS建议EBD注射需要耐心和实践，以达到一致和可靠的结果。 总之，我们描述进行EBD分析斑马鱼幼体实际和直接的方法。迄今为止，使用斑马鱼作为模型系统，尤其是作为人类疾病模型，已迅速扩大。这种扩张部分是由于改善后的斑马鱼系统的当前优势的实验技术的不断发展和改进。该EBD注射技术提供了一个额外的，功能强大的工具，以研究人员的武器库的验证和斑马鱼肌肉疾病模型的研究。这种技术的不断执行和修改有帮助发现新的治疗策略，以及引起疾病机制的潜力。

Muscular dystrophies constitute a group of prevalent and heterogeneous human muscle diseases with specific histopathological features1,2. Symptoms typically associated with this devastating group of diseases include muscle weakness, muscle degeneration, loss of motility, and early mortality1,3. The primary pathomechanisms of muscular dystrophies are the loss of proteins that stabilize the sarcolemma, anchor transmembrane complexes, and mediate cell signaling across the membrane4-6. For example, complete loss of the protein dystrophin, a primary scaffold protein of the dystrophin-glycoprotein complex, results in destabilization of the muscle membrane in Duchenne muscular dystrophy7. Due to the fact that most muscular dystrophies result from mutations in proteins that participate in the link between the extracellular matrix and the sarcolemmal cytoskeleton, a common observation at the cellular level is the loss of sacrolemmal integrity8,9. This understanding of the primary pathomechanism(s) associated with muscular dystrophies is the product of numerous years of research employing animal model systems2,10-15. However, despite advances in the field, there are still limited therapeutic options for treatment or management of the range of dystrophy subtypes. This limitation of viable therapies is due to several key factors: 1) the difficulty of gene therapy strategies, 2) a high frequency of de-novo disease cases and the corresponding lack of translatable animal models, and 3) the lack of rigorous strategies to test the physiological consequences of putative therapeutic agents with clear and reproducible outcome measures.
To overcome some of these limitations, numerous labs including our own are employing zebrafish as a system to model and study human neuromuscular diseases2. To date, zebrafish have proven a valuable tool in disease research. The zebrafish model has been used to identify and validate novel human disease causing mutations16,17, elucidate uncharacterized disease causing mechanisms17,18, and identify novel therapeutic strategies12,19. These advances were made, in part, by the canonical strengths of the zebrafish system such as their optical clarity, ease of genetic manipulation, and ability to breed in large numbers20. Zebrafish have additionally proven amendable to large-scale drug screens21, a valuable method for the identification of novel therapeutics22-24. Regarding muscle disease research, these strengths are complemented by the ability to isolate single zebrafish skeletal muscle fibers via dissociation25 and by the ability to examine myofiber organization in vivo using the optical phenomenon called birefringence26, which collectively allows for rapid determination of macroscopic muscle integrity. Regardless of these available utilities, further tool development is continuously required to advance investigation.
We, and others, have adapted a protocol for EBD injection and analysis in the zebrafish model. EBD is a vital, cell permeable dye that is taken up by damaged, degenerating, or apoptotic cells and then visualized under fluorescence27. To date, EBD analysis has extensively been used to analyze muscle membrane integrity in mouse models of skeletal muscle and heart diseases8,9,27. However, in mammalian preparations, harvested muscle typically requires laborious sectioning or dissection prior to analysis. In zebrafish, direct analysis is possible in high numbers using live and intact animals. In this video article, we will demonstrate the methodology to perform EBD injection and analysis in live zebrafish larvae, with representative images of EBD uptake in the zebrafish dystrophy mutant line sapje15,28. Furthermore, we present a co-injection strategy that allows for increased quantification of EBD preparations.

<table border="0" cellpadding="0" cellspacing="0" width="801">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Fluorescein isothiocyanate-dextran MW 10,000</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">FD10S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Evan&#39;s Blue Dye</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">E2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">A5040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">100 mm Petri dish</td>
			<td style="width:173px;">Fischerbrand</td>
			<td style="width:140px;">FB0875712</td>
			<td>Injection mold base</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">Thin wall glass capillaries</td>
			<td style="width:173px;">World Precision Instruments</td>
			<td style="width:140px;">TW100F-4</td>
			<td>For Injection needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Agarose</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">AGA001</td>
			<td>Injection mold</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Microinjection mold</td>
			<td style="width:173px;">Adaptive Science Tools</td>
			<td style="width:140px;">TU-1</td>
			<td>Injection mold</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Sodium chloride</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">SOD001</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Potassium chloride</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">POC888</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Magnessium chloride hexahydrate</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">M2670</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Sodium phosphate monobasic monohydrate</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">S9638</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">HEPES</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">H4034</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Glucose</td>
			<td style="width:173px;">BioBasic</td>
			<td style="width:140px;">GB0219</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Calcium chloride</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">C1061</td>
			<td>Ringer&#39;s solution</td>
		</tr>
	</tbody>
</table>

analysis of zebrafish larvae skeletal muscle integrity with evans blue dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

所述EBD注射混合物在3旦注入的sapje纯合突变体和野生型同胞的CCV。注射弥漫于心脏腔室（图1B），然后成功注射通过可视化FITC-葡聚糖在根据绿色荧光脉管（图2）进行分析。 经过4小时的潜伏期，EBD摄取检查在用荧光显微镜体节的水平。野生型同胞内表现出任何可见的肌纤维（图3A）没有EBD荧光，而纯合sapje突变体显示EBD摄取，指示损坏肌膜15（图3B）。 <img alt="图1" src="/files/ftp_upload/53183/53183fig1.jpg" /> 图1.注射EBD注入搅拌成斑马鱼安莉芳的共同主静脉（CCV）O操作。 （一）未注射胚胎。箭头表示的CCV注射理想地点。 （二）成功注入CCV。染料进入心脏腔室（箭头），并开始通过脉管被泵送。 （C）不成功的CCV注入将导致部分或全部进入胚胎（箭头）的卵黄囊的染料。 <a href="https://www.jove.com/files/ftp_upload/53183/53183fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53183/53183fig2.jpg" /> 图2.胚胎可以通过观察在绿色荧光FITC-葡聚糖分布于整个脉管紧跟的喷射和前EBD摄取被排序为成功的注射。请点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53183/53183fig3.jpg" /> 图3：EBD将采取由受损的膜纤维（A）野生型的兄弟姐妹在展示肌肉纤维没有EBD荧光。 （二）Sapje ​​纯合突变与多个肌纤维（箭头）内EBD荧光。所有幼虫注射了EBD注射混合，并在3旦一个4小时的潜伏期后进行分析。兄弟姐妹和突变体是由前CCV注射肌纤维脱离排序。 <a href="https://www.jove.com/files/ftp_upload/53183/53183fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye at JoVE.com

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Research

JoVE Journal

Developmental Biology

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

科研

发育生物学

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

通过注射心脏毒素急性骨骼肌再生的诱导

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

斑马鱼幼虫显微注射和成像的微装置

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

慢性刺激在收缩活动性大鼠骨骼肌表型适应性研究中的应用

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

从骨骼肌活检程序分离、培养、表征和分化人类肌肉祖细胞

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

在免疫缺陷小鼠中表演人类骨骼肌异种移植物

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

小鼠骨骼肌肉外泄原发性肌基器的分离和分化

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

斑马鱼胚胎和幼虫的整个山免疫性化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

检查斑马鱼肌肉疾病模型中的肌肉再生

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...