Name: dissection and culture of mouse dopaminergic and striatal explants in three-dimensional collagen matrix assays
Uploaded: 2012-03-23T12:00:00
Description: Watch this Scientific Journal Video about Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays at JoVE.com

胶原基质试验已制定和完善了许多不同的研究小组在过​​去的二，三十年的工作。多巴胺和纹状体植这里描述的方法，从这些研究中大大受益。此外，要感谢她的帮助下设立纹状体植文化Asheeta普拉萨德。在实验室工作是由人类前沿科学计划组织（职业发展奖），荷兰健康研究与发展组织（ZonMW VIDI和弦ZonMW），Europanian联盟（mdDA NeuroDev，FP7/2007-2011的格兰特222999）（RJP），荷兰科学研究组织（TopTalent; ERES）时。

1。鼠尾胶原的制备<ol><li>收集6-10成年鼠尾（这是可能的尾巴，直到使用储存在-20°C）。 </li><li>隔夜浸泡在95％乙醇的尾巴在室温（RT）。 </li></ol> 解剖老鼠尾巴（组织培养罩）： （保持在70％乙醇的工具时，不使用他们，并确保在整个过程中使用的所有解决方案，工具和玻璃器皿是无菌） <ol start="3"><li>收集从尾巴肌腱，切断尾巴尖。同一个钳子的大尾巴结束。?...

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这里描述的胶原基质试验已在过去的几十年里许多不同的实验室使用和改进探讨轴突导向分子和神经系统的各种（例如，见参考文献5-8）。这些研究表明，这种试验是研究轴突导向分子不同（中级）靶组织分泌的影响和调控的有力工具。 然而，应当指出，胶原基质是外环境（ECM）的替代品，但显然缺乏通常在ECM目前许多蛋白质​​。 ECM的组件被称为影响轴突导向分子10</s...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>胎牛血清</td><td> BioWhittaker </td><td> 14-801F </td><td></td></tr><tr><td>谷氨酰胺（200MM） </td><td>临时机场管理局</td><td> M11-004 </td><td></td></tr><tr><td> HEPES </td><td> VWR国际</td><td> 441476L </td><td></td></tr><tr><td> β-巯基乙醇</td><td>默克公司</td><td> 444203 </td><td></td></tr><tr><td>最低（MEM）的主要媒介</td><td> Gibco公司</td><td> 61100-087 </td><td></td></tr><tr><td>的Neurobasal </td><td> Gibco公司</td><td> 21103-049 </td><td></td></tr><tr><td> B27的</td><td> Gibco公司</td><td> 17504-044 </td><td></td></tr><tr><td>莱博维茨的L-15中</td><td> Gibco公司</td><td&gt; 11415-049 </td><td></td></tr><tr><td>青霉素链霉素</td><td> Gibco公司</td><td> 15070-063 </td><td></td></tr><tr><td>延长黄金抗淬灭试剂</td><td> Invitrogen公司</td><td> P36930 </td><td></td></tr><tr><td>透析管</td><td>光谱实验室</td><td> 132660 </td><td></td></tr><tr><td>兔抗酪氨酸羟化酶</td><td> PEL-FREEZ </td><td> P40101-0 </td><td></td></tr><tr><td>兔抗Darpp32（H 62） </td><td>圣 - 克鲁兹</td><td> SC-11365 </td><td></td></tr><tr><td>鼠抗βIII微管蛋白</td><td>西格玛</td><td> T8660 </td><td></td></tr><tr><td>的Alexa Fluor标记的二抗</td><td> Invitrogen公司</td><td></td><td></td></tr></tbody></table>

dissection and culture of mouse dopaminergic and striatal explants in three-dimensional collagen matrix assays

中脑多巴胺（mdDA）神经元的项目，通过对几个领域，在端脑，包括纹状体内侧前脑束。同样地，中等刺在纹状体的神经元，引起纹状体黑（直接）通路支配黑质2。的这些轴突大片的发展是依赖过多的轴突生长和指导线索，包括由突起或释放（中间）目标地区3,4分子组合行动。这些可溶性因子可以在体外研究的培养mdDA和/或在胶原基质，它提供了一个立体的模仿外环境的轴突基板纹状体植。此外，胶原基质允许放置在附近（例如，见参考文献5和6）其他植或细胞蛋白形成相对稳定的梯度。在这里，我们描述的PUR的方法ification的鼠尾胶原蛋白，多巴胺和纹状体外植体的显微切割，他们的文化，在胶原蛋白凝胶和随后的免疫组化和定量分析。首先，E14.5小鼠胚胎的大脑是孤立和多巴胺和显微纹状体植。这些植（CO）盖玻片上培养48至72小时在体外在胶原凝胶。随后，轴突的预测是使用可视化的神经标记（如酪氨酸羟化酶，DARPP32，或βIII微管蛋白）和轴突生长和吸引力或排斥力的轴突反应进行量化。这神经的编制是一项有用的工具， 在的mesostriatal与纹状体黑轴突生长和发育过程中的指导体外培养的细胞和分子机制研究。采用此法，它也有可能评估其他（中级）多巴胺和纹状体轴突的目标，或测试特定的分子线索。

Watch this Scientific Journal Video about Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays at JoVE.com

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

中脑多巴胺系统及纹状体植为胶原基质检测<em&gt;在体外</em&gt;分析的mesostriatal和纹状体黑通路发展。轴突生长和指导，在此法可以操纵和量化。它也可以被修改为评估其他地区或分子线索。

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