Name: visualizing the live drosophila glial-neuromuscular junction with fluorescent dyes
Uploaded: 2009-05-13T00:00:00
Description: Watch this Scientific Journal Video about Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes at JoVE.com

这个项目是由CIHR和NSERC。我们要感谢创造表达DsRed的标记SSR（北京）和不列颠哥伦比亚大学生物成像设施的飞株倒钩Jusiak。

第1部分：组织准备<ol><li>我们的目标是组织编制的神经系统是完整的蝇蛆，但体壁肌肉的内表面接触到一个人工血淋巴，可定位接近一个良好的光学显微镜盖玻片。换句话说，里面出幼虫准备。 由于传统的组织筹备工作涉及切割，固定和体壁的肌肉伸展，有时消除神经系统的一部分，我们需要一种不同的方法。 我们希望内而外的动物，因为我们想获得以上时间在突触的结构特...

<ol>
	<li>Macleod, G. T., Marin, L., Charlton, M., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+Vesicles:+Test+for+a+role+in+presynaptic+Calcium+regulation.">Synaptic Vesicles: Test for a role in presynaptic Calcium regulation.</a> J. Neurosci. 24, 2496-2505 (2004).</li><li>Macleod, G. T., Hegstro, M., Wojtowicz, M., Charlton, M. P., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Calcium+signals+in+Drosophila+motor+neuron+terminals.">Fast Calcium signals in Drosophila motor neuron terminals.</a> J. Neurophysiology. 88, 2659-2663 (2002).</li><li>Morales, M., Ferrus, A., Martinez-Padron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+calcium-channel+currents+in+normal+and+csp+mutant+Drosophila+peptidergic+terminals.">Presynaptic calcium-channel currents in normal and csp mutant Drosophila peptidergic terminals.</a> Eur J Neurosci. 11, 1818-1826 (1999).</li><li>Stork, T., Engelen, D., Krudewig, A., Silies, M., Bainton, R. J., Kla&#168;mbt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+Function+of+the+Blood&#8211;Brain+Barrier+in+Drosophila.">Organization and Function of the Blood&#8211;Brain Barrier in Drosophila.</a> J. Neurosci. 28, 587-597 (2008).</li></ol>

此过程允许长期住标记的蛋白和细胞进程的成像。 原位组织准备，我们描述了一个完整的和运作的中枢神经系统，百佳和反射电路。该组织准备已超过标准的幼虫蝇肌肉协议，幼虫体壁的肌肉被拉伸（当它被固定）优势。拉伸可以扭曲突触形态和触发反射的基础收缩。我们出预习内机械性能稳定，并且有特别好的光学，促进细胞的变化实时高分辨率分析。此外里出来准备存活一个小时，并...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>HL-6: Artifical Drosophila hemolymph, with 5 mM L-glutamate added, and 2 mM Calcium.</td>
<td>Reagent</td>
<td>NA</td>
<td>NA</td>
<td>5 mM L-glutamate blocked muscle contractions. We used Molecular grade L-Glutamate (Sigma).
2 mM Calcium is close to physiological Calcium levels in natural larval hemolymph.
References: Macleod et al 2002 and Macleod 2004
</td>
</tr>
<tr>
<td>Dextran, Alex Fluor 680; 10,000 MW, anionic, fixable</td>
<td>Reagent</td>
<td>Molecular Probes/Invitrogen</td>
<td>D34680</td>
<td>Use a small volume perfusion chamber to keep the total volume of dye low</td>
</tr>
<tr>
<td>Anti-HRP-CY5 conjugate (goat)</td>
<td>Reagent</td>
<td>Jackson ImmunoResearc</td>
<td>123-175-021</td>
<td>Dilute 2.0 mg into 1 ml ddH2O; aliquot into 4 microliter aliquots. Freeze at &#x2013;20C. Dilute one aliquot into 100 microliters of HL-6</td>
</tr>
<tr>
<td>Alexa 647 antibody labeling kit</td>
<td>Reagent</td>
<td>Molecular Probes/Invitrogen</td>
<td>A10475</td>
<td>We prepared a total of 80 micro liters of conjugated primary antibody, and stored as 2 microliter aliquots. We diluted each aliquot into 100 microliter of HL-6 for labeling.</td>
</tr>
<tr>
<td>Custom Formulated Objective Oil, refractive index 1.3379</td>
<td>Reagent</td>
<td>Cargill Labs</td>
<td>Custom Formulated</td>
<td>&nbsp;</td>
<tr>
<td>Ultra Fine Forceps</td>
<td>Tool</td>
<td>Fine Science Tolls</td>
<td>11252-23 or 11295-20</td>
<td>&nbsp;</td>
<tr>
<td>Spring scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>91500-09</td>
<td>&nbsp;</td>
<tr>
<td>Ultra fine clipper scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>15200-00</td>
<td>&nbsp;</td>
<tr>
<td>Perfusion Chamber RC 20 Series</td>
<td>Tool</td>
<td>Warner Instruments</td>
<td>64-02222</td>
<td>&nbsp;</td>
<tr>
<td>Spinning Disc confocal</td>
<td>Microscope</td>
<td>Quorum</td>
<td>Quorum Wave FX</td>
<td>Mounted on a Leica DMI6000 Inverted Microscope</td>
</tr>		</tbody>
		</table>
		</div>

visualizing the live drosophila glial-neuromuscular junction with fluorescent dyes

我们的项目确定在发展中的幼虫飞神经肌肉突触的绿色荧光蛋白标记的胶质结构。为了看住胶质细胞神经​​肌肉突触的发展，我们开发了一个幼虫的组织编制了完整的幼虫生活的功能，但也有良好的光学性能。这个新的编制也不允许灌流液进入突触。我们用蝇蛆，沉浸在人工血淋巴，寒心他们放宽了其正常的有节奏的身体收缩。下一步，我们解剖了每个动物的后段，并用钝器昆虫针推通过体腔口部位落后。这外翻幼虫体壁，就像把袜子内而外。我们完成了超细清扫剪刀剥离，从而暴露了体壁肌肉内脏的一面。在NMJ的胶质结构对膜有针对性的绿色荧光蛋白，胶质细胞特异性启动子的控制下。突触后膜，肌肉中的SSR（Subsynaptic Reticula）表示突触针对性DsRed的。我们需要清楚地标记的运动神经元终端突触的第三部分。要做到这一点，我们采用的主要抗体，以辣根过氧化物酶结合到远红外发光flurophore。 perisynaptic空间之间的运动神经元终端和SSR的染料扩散性能测试，我们采用一个大型的葡聚糖分子的共轭远红外发光flurophore和收集到的图像解决方案。

Watch this Scientific Journal Video about Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes at JoVE.com

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

我们描述的神经胶质细胞神经​​肌肉突触的结构特点，以一种新颖的由内而外的活飞使用共聚焦显微镜荧光染料的幼虫的组织准备。我们辣根过氧化物酶的荧光抗体标记的活神经元的终端，也可视荧光右旋糖酐perisynaptic空间。

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Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

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Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

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