Name: laser-inflicted injury of zebrafish embryonic skeletal muscle
Uploaded: 2013-01-30T12:00:00
Description: Watch this Scientific Journal Video about Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle at JoVE.com

我们感谢鲍勃·诺瓦克（安道尔的技术）的技术帮助和建议。 SA-S。的德意志研究联合会（DFG）的海森堡奖学金的支持。这项工作是支持，DFG授予SE2016/7-1。

1。单细胞进行标记注入单细胞阶段的胚胎的质粒编码GFP或任何GFP融合蛋白的β-肌动蛋白启动子的控制下。在开发过程中，GFP的表达，在时尚的马赛克。这里，我们使用的转基因构建体的Tg []，放置的α-辅肌动蛋白-GFP融合蛋白的β-肌动蛋白启动子的控制下的β-肌动蛋白：α-辅肌动蛋白-GFP。 2。胚胎嵌入<ol class="lalpha"><li>收藏报错斑马鱼胚胎中...

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激光介导的损伤是造成所需大小的伤口，切除细胞，以研究在受控条件下的斑马鱼胚胎再生一个功能强大的方法。值得注意的是，细胞可以有针对性的精确（ 图2），可以控制损伤区以及定时。随后，上述损伤部位和再生过程很容易监测，记录（ 图3），并进行分析（ 图1）。然而，虽然激光以及集中在x-和y-轴，它不能完全集中在z-轴。这可能会导致损坏位于?...

<table border="1"><tbody><tr><td>加热块</td><td></td><td></td><td></td></tr><tr><td>对＃5镊子</td><td>杜蒙</td><td></td><td></td></tr><tr><td>载玻片</td><td>门泽尔</td><td></td><td> 76 x 26毫米</td></tr><tr><td>盖玻片</td><td>罗斯</td><td></td><td> 50×24毫米＃1 </td></tr><tr><td>凡士林</td><td></td><td></td><td></td></tr><tr><td>体视显微镜</td><td>徕卡</td><td></td><td> MZFLIII </td></tr><tr><td>微点激光</td><td>安道​​尔技术</td><td></td><td></td></tr><tr><td>荧光显微镜</td><td>蔡司</td><td></td><td> Axioplan II </td></tr><tr><td> Metamorph软件</td><td>分子器件</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 试剂 <ul><li>低熔点琼脂糖（＃50081，龙沙） </li><li>三卡因储备溶液：400毫克三卡因（＃A-5040，Sigma-Aldrich公司）/ 100毫升蒸馏水2 O pH值9.0 </li><li> E3介质（5 mM氯化钠，0.17 mM KCl中，0.33毫的CaCl 2，0.33毫用MgSO 4） </li></ul></td></tr></tbody></table>

laser-inflicted injury of zebrafish embryonic skeletal muscle

不同的实验方法，使用鼠标的目的是研究肌肉再生，的包括myotoxin注射（布比卡因，心脏毒素或notexin），，，肌肉移植（神经损伤周围血管离断术诱导再生），大强度运动引起肌肉损伤，但也小鼠的肌营养不良症模型如评论这些方法mdx小鼠（参见图1）。在斑马鱼基因的方法包括突变体，表现出肌营养不良症的表型（，如runzel 2或sapje 3）和反义寡核苷酸吗啉代，阻止营养不良相关基因的表达。此外，化学方法，也有可能， 例如 ，加兰他敏，一种化学化合物抑制乙酰胆碱酯酶，从而导致hypercontraction，最终导致肌营养不良症5。然而，遗传学和药理学方法克enerally影响个人所有的肌肉内，而身体造成伤害的程度，更容易控制时间和空间上1。本地化的人身伤害可以作为内部控制的评估对侧肌肉。事实上，我们最近用激光消融介导的细胞研究在斑马鱼胚胎6骨骼肌再生，而另一组最近报道了利用双光子激光（822 nm）的损害本地个别胚胎斑马鱼肌肉的细胞膜细胞7。 在这里，我们报告使用微点激光（安道尔的技术）在斑马鱼胚胎骨骼肌细胞损伤的方法。微点激光是一种高能量的激光，这是适合于在波长为435nm的靶细胞消融。的激光以这样的方式被连接到显微镜（在我们的设置，光学显微镜从蔡司）显微镜，可以使用在同一时间f或聚焦在样品上的激光和用于可视化的伤人（明或荧光）的影响。用于控制激光脉冲的参数包括波长，强度和脉冲数。 由于其透明度和外部的胚胎发育，斑马鱼胚胎的研究为激光损伤和随后的复苏是非常适合的。受精后1至2天，体节骨骼肌细胞逐渐接受由前向后成熟体节发育的进展，从树干尾8，9。在这些阶段，胚胎自发地抽搐，并开始游泳。最近斑马鱼已被确认为一个重要的脊椎动物模式生物组织再生的研究，许多类型的组织（心脏，神经，血管等）损伤的成年斑马鱼10，11后是可以再生的。

激光介导的损伤进行固定1日龄胚胎。数的激光脉冲在图1中所示，可以生成一个小伤口，由损坏的，盘绕，肌动蛋白丰富的肌原纤维，通常拉伸之间的体节边界容易辨认。然而，较高的激光脉冲造成的大规模破坏的体节块，其中最肌原纤维被破坏。不过，我们可以从字面上遵守“时间可以治愈一切伤口”愈合速度比大的小伤。恢复后，肌动蛋白，肌原纤维染色显示再次跨越正常的体节...

Watch this Scientific Journal Video about Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle at JoVE.com

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

这里介绍的方法，包括精确的具有高能量的激光脉冲和随后的分析随时间的伤害和其恢复损伤的活斑马鱼胚胎。我们还展示了如何转基因标记的单或骨骼肌细胞可以被跟踪和激光损伤后。

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 ...

Research

JoVE Journal

Biology

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 ...

科研

生物学

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 ...

Mitochondrial Isolation from Skeletal Muscle

骨骼肌的线粒体分离

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

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 ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

体外评估对离体骨骼肌的收缩，疲劳性和交替“

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

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 ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...