Name: analysis of embryonic and larval zebrafish skeletal myofibers from dissociated preparations
Uploaded: 2013-11-13T19:30:00
Description: Watch this Scientific Journal Video about Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations at JoVE.com

作者要感谢道林实验室的成员（亚伦Reifler，特伦特沃，安吉拉布斯塔，和威廉特尔弗）​​促成了技术的发展和生产的手稿。这项工作是由该研究所陶布曼，小儿科的部门在密歇根大学，并在部分从肌肉萎缩症协会（JJD MDA186999）和美国国立卫生研究院（JJD 1K08AR054835）资助。

1。聚-L-赖氨酸的制备涂层盖玻片（时间：1小时） 涂层盖玻片允许快速肌纤维的沉降和附着。这可能在肌纤维隔离（下面的步骤2）的解离步骤中进行。 <ol><li>切割和封口膜放置在60毫米培养皿（任何品牌）的底部。 </li><li>放置显微镜盖玻璃卡瓦（12毫米直径）上的Parafilm在60mm组织培养皿或单独放置在单个井的24孔板中。 </li></ol>注1：这些小圆形盖玻片有助?...

<ol>
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斑马鱼是研究体内 25,27,28肌肉发育和功能的有力脊椎动物模型系统。他们也成为一笔宝贵的财富用于模拟人类肌肉疾病14,15,20,29。虽然大踏步已采取提前斑马鱼的肌肉功能和肌肉疾病的研究的使用和应用，有一个恒定的迫切需要开发工具，让更多的深入分析，赞美的遗传，形态，行为和功能优点斑马鱼已经提供17。因此，我们已经适应，并开发了一个简单和强大的技...

骨骼肌是负责生成所必需的蠕动收缩力高度专业化的组织。收缩是通过称为兴奋-收缩（欧共体）耦合，转换电信号从细胞内贮存1,2钙释放的过程开始。细胞内钙释放激活的肌节缩短和产生力量。负责调解神经肌肉接头处传递3，EC耦合​​4,5，和肌动蛋白-肌球蛋白依赖性收缩6的分子机器的许多具体的组件继续深入研究的持续主题。此外，在收缩7,8和介导的肌纤维和细胞外基质7,9之间的信令稳定肌细胞膜蛋白已经确定，并研究了很详细。 在许多基因的肌肉结构的重要突变ND功能已被确定为人体骨骼肌肉疾病的原因（ <a href="http://www.musclegenetable.org/" target="_blank">http://www.musclegenetable.org/</a> ）。这些疾病，大致分类为基础的临床和病理特征骨骼肌肌病和肌营养不良症，都与肌肉无力，终身残疾，早期死亡率10,11相关联。斑马鱼已被证明是用于建模和研究人类骨骼肌肉疾病8,12,13一个优秀的系统。它已被用来验证新的基因突变8，确定疾病的病理生理学14,15的新内容，并找出新的治疗方法15,16。斑马鱼研究人类肌肉疾病的功率涉及大量的后代，肌肉结构和功能，斑马鱼胚胎的光学透明度，并且易于显影斑纹的遗传药理学和操纵的快速发展的鱼17。 我们和其他人12,18,19最近开发出一种简单的技术为肌纤维来自发展中国家斑马鱼的快速，高效的隔离。这种方法使肌纤维的考试比可以通过全胚胎分析提供更多细节。该技术已被开发用于蛋白质亚细胞定位20以及表征为重要病理特征为在新开发的疾病模型21的验证研究的一部分的识别。此外，孤立的肌纤维还可以用于实时成像和电生理研究22，技术，允许对肌肉功能的关键方面的审讯。的特定协议为肌纤维隔离，随着后续的分析实验的两个例子，在该原稿的其余部分详细说明。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>24-well culture plate</td>
			<td>Corning</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Invitrogen Gibco</td>
			<td>70011</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Independent medium</td>
			<td>Invitrogen Gibco</td>
			<td>18045</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type II</td>
			<td>Worthington Biochemical</td>
			<td>LS004186</td>
			<td>Lot 41H12764</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington Biochemical</td>
			<td>L5004188</td>
			<td></td>
		</tr>
		<tr>
			<td>8% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>322415</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep serum</td>
			<td>Sigma</td>
			<td>S3772</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Fischerbrand</td>
			<td>12-545-82 12CIR-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Sigma</td>
			<td>P5147</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m Filter</td>
			<td>BD Biosciences</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m Filter</td>
			<td>BD Biosciences</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold antifade reagent</td>
			<td>Invitrogen</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#945;-Actinin antibody</td>
			<td>Sigma</td>
			<td>A5044</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-RYR antibody</td>
			<td>Abcam</td>
			<td>34C</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor antibody</td>
			<td>Invitrogen</td>
			<td>A-21425</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>Fischerbrand</td>
			<td>0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Fischerbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Caffeine</td>
			<td>Sigma</td>
			<td>C0750</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of embryonic and larval zebrafish skeletal myofibers from dissociated preparations

斑马鱼已被证明是一个有价值的模型系统研究骨骼肌功能和研究人类肌肉疾病。尽管通过体内分析骨骼肌在斑马鱼提供的，可视化的复杂和精细结构的蛋白质环境负责的肌肉功能，尤其是在整个胚胎中的诸多优点，可能会产生问题。这个障碍源于斑马鱼的骨骼肌（60微米）和肌节的甚至更小尺寸的小尺寸。在这里，我们描述和演示从斑马鱼胚胎和幼虫分离骨骼肌纤维的简单和快速的方法。我们还包括了协议，说明后期制备技术用于分析的肌肉结构和功能非常有用。具体来说，骨骼肌蛋白质我们详细介绍了随后的免疫细胞化学定位，并通过活细胞钙成像刺激钙释放的定性分析。总体来说，这款视频本文提供了斑马鱼的骨骼肌纤维的分离和表征，一种技术，它提供了一种导管，用于肌肉结构和功能的无数的后续研究的直向前和有效的方法。

肌纤维的荧光免疫染色（ 图2） 从显示预期的肌纤维荧光标记图形图像成功分离和电镀后免疫染色。在肌纤维已被标记为与抗兰尼碱受体（1:100）（ 图2A）或抗α-辅肌动蛋白（1:100）（ 图2B）的抗体，并分别揭示三联及Z带的免疫染色。用二抗的Alexa Fluor 555（1:500）。图像利用共聚焦显微镜拍摄的。 从肌纤维...

Watch this Scientific Journal Video about Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations at JoVE.com

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

斑马鱼是用于模拟骨骼肌的人类疾病的一个新兴的系统。我们描述了一个快速，高效的方法从胚胎和幼虫的斑马鱼隔离骨骼肌肌纤维。这种方法产生了高密度的肌纤维制备适合的单骨骼肌纤维形态的研究中，蛋白质的亚细胞定位，以及肌肉生理学。

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 ...

Research

JoVE Journal

Biology

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

科研

生物学

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 Zebrafish Embryonic Eye Tissues

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 ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

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 ...

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 ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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 ...