逆行神经，大港，并用荧光染料的神经根装载
Summary
我们描述了一个简单和低成本的技术引入神经元或神经束，使用聚乙烯吸吸管荧光灯和钙敏感染料的高浓度。
Abstract
逆行标记神经元，是一个标准的解剖方法1,2，也被用来加载到3-6神经元钙和电压敏感染料。一般来说，染料应用于固态晶体或局部压力注射用玻璃吸管。但是，这可能导致稀释染料和减少标签的强度，尤其是当几个小时的染料扩散。在这里，我们展示了一个简单和低成本的技术引入使用聚乙烯吸吸管，充满了染料溶液的神经元的荧光灯和离子敏感的染料。这种方法提供了可靠的方式维持在整个装载过程中的轴突接触高浓度的染料。
Protocol
荧光葡聚糖已被用作解剖工具和成像神经元活动1-4。领域等 。（2009）发表了一份协议，为应用重点是作为一个模型系统的脊髓轴突束离子和电压敏感染料。在这里，我们描述了荧光和/或离子敏感的染料， 在体外 optophysiological和形态学研究申请截止前根，背根或脊髓神经束更详细的过程。 1。 I型和II型移液器开始拉了一个酒精灯的火…
Discussion
<p class="jove_content"我们在这里描述为简单和成本效益的协议，引入神经元，神经和脊髓束的染料。此方法涉及确定的解剖途径暴露的装载过程持续时间为一个高度集中的染料溶液。逆行标记这一点背景与目标站点的结果相比，微量注射，洗澡的应用和电技术。然而，该方法仅限于中枢神经系统的轴突束可以分离的站点。此外，由于染料扩散到沿轴突到达细胞体，加载时间将取决于从染料应用到神经元胞体部位的距?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
<p class="jove_content">支持这项工作是由神经系统疾病的国家机构和国立卫生研究院中风壁间方案。我们还想感谢他的早期贡献的方法，并在图3的数据乔治Mentis博士。</p>
Materials
| Material Name | Company | Catalogue number | Comments | |
| Tubing | PE90 (IDxOD:0.034″x0.050″; Wall Thikness:0.008″) | Clay Adams Brand Intermedic | 427421 | For Type I & Type II pipettes |
| NSF-51(IDxOD:1/16×1/8; Wall Thikness:1/32) | PharMed BPT, Cole-Parmer | AY242002 | For Flexible tubing | |
| Syringe | 1ml insulin Syringe U-100 | Becton Dickenson | 329650 | |
| Needle | 19G x1-1/2″ Metal Hub Needle | MONOJECT | 200136 | Connecting Type II pipette to syringe |
| Alcohol Lamp | ||||
| Stopcock | Three-way Stopcock with Male Luer Slip Adapter | Baxter Healthcare Corp. | 2C6241 | Use with syringe |
| Holder | H-1 electrode holder | Narishige | H-1/12 | |
| Magnet stand | Narishige | GJ-8 | ||
| Micromanipulator | Narishige | M-3333 | ||
| Tweezers | S&T DUMONT Swiss 00632-11 | DUMONT | JF-5 TC | |
*Those are suggested materials. Can be replaced with any compatible products
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Citer Cet Article
Blivis, D., O’Donovan, M. J. Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes. J. Vis. Exp. (62), e4008, doi:10.3791/4008 (2012).