激光捕获显微切割技术的果蝇外周神经元
Summary
在这个视频文章中，我们目前的方法隔离单个或多个<em>果蝇</em> DA神经元使用红外线（IR）的捕获激光捕获显微切割（LCM）的类三龄幼虫。从孤立的神经元获得的RNA可以很容易地用于下游应用，包括QRT – PCR或芯片分析。
Abstract
树突状树枝状（DA）神经元的果蝇周围神经系统（PNS）在调查的基础类特定的树突形态发生 1,2的分子机制提供一个极好的的模型系统。为了方便类特定的DA神经元发展的分子生物学分析，至关重要的是在一个纯粹的人口获得这些细胞。虽然一系列不同的细胞和组织特异性的RNA分离技术中存在的果蝇细胞，包括基于磁珠细胞净化3,4，荧光激活细胞分选（FACS）5-8，和RNA结合蛋白为基础的战略9 ，概无这些方法可以很容易地利用空间精度与高度隔离的单个或多个类特定的果蝇DA能神经元。激光捕获显微切割（LCM）已成为一个非常强大的工具，可以用来从组织切片中分离出特定的细胞类型，具有高度的空间分辨率和精度。然后，可以从分离的细胞获得的RNA用于包括在一个给定的细胞类型10-16的QRT – PCR和基因芯片表达谱分析。迄今为止，液晶显示模块没有被广泛应用在果蝇的组织和细胞17,18，包括发展第三龄幼虫阶段的DA能神经元的分析。
在这里，我们目前对果蝇的大神经元使用的红外（IR）类LCM的隔离，我们优化的协议。这种方法允许单一，类特定或多个的DA能神经元具有高特异性和空间分辨率的捕捉。年龄相匹配的三龄幼虫表达的UAS – mCD8：绿色荧光蛋白 19基因的控制下的IV级DA神经元具体PPK – GAL4 20驱动程序或泛DA神经元特异性21 – 7 – GAL4 21驱动程序是用于这些实验。从孤立的DA能神经元获得的RNA是非常高的质量，并可以直接用于下游应用，包括QRT – PCR或芯片分析。此外，这种液晶显示模块协议，可以方便地用于捕捉到其他果蝇的细胞类型的不同阶段的发展依赖于具体，GAL4驱动 GFP的表达模式的细胞类型的。
Protocol
对果蝇 LCM的外周神经元的一般意见允许来自6个小时，一个星期或液晶显示模块不再取决于组织类型和所需的细胞数量。 所有的程序都进行严格的无RNase条件以下的标准程序。幼虫表达无论是21 – 7 – GAL4，无人机mCD8：：GFP或PPK – GAL4 UAS – mCD8：：GFP转基因记者线，这些实验中使用。 1。准备幼虫收集30-40年龄相?…
Discussion
本协议描述了我们对果蝇通过LCM的外周神经元的隔离的优化方法。虽然这LCM协议旨在为特定的隔离单，类特定或多个果 蝇的大神经元从3龄幼虫阶段的发展，稍作修改的协议可以很容易地适用于其他果蝇的细胞类型的捕捉从所有发展使用不同的GAL4的阶段 ， 记者UAS – mCD8 – GFP转基因标签的细胞类型或类型的利益。我们的研究结果表明，激光捕获微解剖DA?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢博士。裕农扬和韦斯Grueber提供援助，带LCM飞的股票，在本研究中使用的，弗吉尼亚Espina，伊曼纽尔Petricoin博士和博士兰斯利奥塔。作者承认这项研究的支持（DNC）和乔治梅森大学教务长办公室（EPRI）的托马斯F和凯特米勒Jeffress纪念信托。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| 10X Phosphate Buffered Saline (PBS) | MP Bioproducts | PBS10X02 | Diluted to 1X working solution | |
| 2.5% Trypsin | Sigma-Aldrich | T1426 | ||
| RNase-AWAY | Sigma-Aldrich | 83931 | ||
| Xylenes, histological grade | Fisher Scientific | X3S-4 | ||
| RNAse-free water | Fisher Scientific | BP561-1 | ||
| Optimal Cutting Temperature (OCT) compound | Tissue-Tek | 4583 | ||
| PicoPure RNA Isolation Kit | Molecular Devices | KIT0204 | Follow manufacturer’s instructions | |
| Thin walled reaction tube with domed cap | GeneAmp, Applied Biosystems | N8010611 | ||
| ExtracSure Sample Extraction Device | Molecular Devices | LCM 0208 | ||
| Incubation Block for sample extraction from CapSure HS Caps | Molecular Devices | LCM0505 | ||
| Alignment Tray for CapSure HS LCM Caps and ExtracSure Sample Extraction Devices | Molecular Devices | LCM0504 | ||
| CapSure HS LCM caps | Molecular Devices | LCM0213 | ||
| 75×100 mm glass slides | Fisher Scientific | 12-544-3 | ||
| Tissue embedding molds | Fisher Scientific | NC9642669 | ||
| Polypropylene pestle for 1.5 ml microcentrifuge tubes | USA Scientific | 1415-5390 | ||
| 70% Ethanol; 95% Ethanol; 100% Ethanol | ||||
| Dry ice |
Equipment:
- Cryostat
- 50 ml conical tube for slide fixation, rinsing, trypsin treatment and ethanol/xylene dehydration
- -80°C freezer
- Incubator
- PixCell IIe LCM Instrument with Fluor 300 epifluorescence optics optimized for EGFP (Molecular Devices-Molecular Devices)
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Tags
Laser Capture MicrodissectionDrosophila Peripheral NeuronsDendritic ArborizationDa NeuronsMolecular MechanismsClass-specific Dendrite MorphogenesisRNA Isolation TechniquesMagnetic Bead-based Cell PurificationFluorescent Activated Cell Sorting (FACS)RNA Binding Protein-based StrategiesLaser Capture Microdissection (LCM)Spatial PrecisionQRT-PCRMicroarray Expression ProfilingThird Instar Larval Stage
Citer Cet Article
Iyer, E. P. R., Cox, D. N. Laser Capture Microdissection of Drosophila Peripheral Neurons. J. Vis. Exp. (39), e2016, doi:10.3791/2016 (2010).