Name: automated quantification of synaptic fluorescence in c. elegans
Uploaded: 2012-08-10T12:00:00
Description: Watch this Scientific Journal Video about Automated Quantification of Synaptic Fluorescence in C. elegans at JoVE.com

笔者想感谢贝纳姆A.协助协议的发展。这项工作是由NIH资助NS06747 BAB

1。成像蠕虫的制备这部分协议的基础上发表C.线虫培养技术1,2， 图1中概述。 <ol><li>生长在高的蛋白胨NGM的琼脂板（10厘米）播种与NA22 ... E.蠕虫大肠杆菌的细菌，直到近饿死。染色，如果一个板块将可靠地生产出足够的蠕虫1个人污点，一路上，考虑损失和用于选择成像蠕虫（见第2部分）的严格标准。 </li><li> DDH 2 0几毫升倾?...

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这里介绍的方法设计到突触的大量人口在 C中提取的多参数的定量数据线虫 ，同时最大限度地提高治疗组内的一致性。三个特点有助于实现这些目标。首先，免疫组化进行同步蠕虫种群，以确保所有的动物都是一样的年龄。这一步是至关重要的，因为发育调控的表达水平可能掩盖的实验性治疗的效果（如UNC的49 GABA受体免疫变化的若干倍在发展（光戴维斯等人，在准备中））。...

<table border="1"><tbody><tr><td> 该软件的名称 </td><td> 公司 </td><td></td><td> 评论（可选） </td></tr><tr><td> volocity V4.0或更高</td><td>珀金埃尔默/ Improvision </td><td></td><td>检查你访问这个软件的本地影像核心设施。演示软件可在珀金埃尔默网站。这种方法只需要Volocity定量模块。 </td></tr><tr><td></td><td></td><td></td><td> 表2。特定的试剂和设备。 </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 鸡蛋缓冲区 </td><td>氯化钠 氯化钾 氯化钙 MgCl 2的 肝素钠 （调整pH值至7.3） </td> &lt;TD&gt; 118毫米 48毫米 2毫米 2毫米 24毫米</td></tr><tr><td> 碱性次氯酸钠（5毫升） </td><td>新鲜的家用漂白水（丢弃后30天内开瓶） 10N的氢氧化钠 DDH 2 O的 </td><td> 1.0毫升0.25毫升 3.75毫升</td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td> 表1。解决方案。 </td></tr></tbody></table>

automated quantification of synaptic fluorescence in c. elegans

突触的强度是指突触突触前神经递质释放的事件的反应幅度，并有一个整体的神经电路功能产生重大影响。突触强度严重依赖于大量聚集在突触的突触后膜部位的神经递质受体。受体水平的发展，建立和受体之间贩运表面本地化，突触，细胞内池代表突触可塑性和神经调节的重要机制，可以通过改变。严格的方法来量化本地化的突触神经递质受体的丰度是必不可少的，以研究突触的发展和可塑性。荧光显微镜是一个最佳的方法，因为它保留了空间信息，区分从非突触池的突触，和本地化为不同类型的突触受体人口之间的歧视。遗传模式生物线虫elegans是特别适合这些研究中，由于体积小，相对简单的神经系统，其透明度，并提供强大的基因技术，使本地完整的动物突触检查。 在这里，我们提出了一个定量荧光标记C.突触神经递质受体的方法线虫 。其主要特点是多面的共聚焦显微镜输出文件，制表位置，体积，荧光强度，并为每个突触的总荧光三个维度的单个突触的自动识别和分析。这种方法有两个超过手册z平面聚焦数据预测分析的主要优点。首先，因为每一个聚焦数据集平面，没有数据丢失通过Z-平面上的投影，通常是基于像素的强度平均值或最大值。其次，识别突触是自动化的，但可以通过前检查perimenter作为数据分析所得，允许快速，准确地从大量的突触中提取数据。数百数千突触每个样品可以很容易地获得，产生大量的数据集，以最大限度地提高统计能力。注意事项准备C。进行分析，并进行聚焦成像，以尽量减少动物在治疗组之间的变异线虫进行了讨论。尽管分析C。线虫突触后受体，这种方法通常是有用任何突触本地化的蛋白质，事实上，任何荧光信号，定位于离散集群，puncta，或细胞器的类型。 执行过程中的三个步骤：1）制备样品，2）聚焦成像，和3）图像分析。具体到 C步骤1和2 线虫 ，而第3步是普遍适用于任何点状共聚焦显微镜的荧光信号。

Watch this Scientific Journal Video about Automated Quantification of Synaptic Fluorescence in C. elegans at JoVE.com

Automated Quantification of Synaptic Fluorescence in C. elegans

强烈的大量聚集在突触的神经递质受体影响突触强度。这种方法量化单突触决议的荧光标记的神经递质受体在三个层面<em&gt; C。线虫</em&gt;，让数百个突触被迅速的特点是在一个单一的样本没有推出Z-平面上的投影的扭曲。

突触荧光自动定量<em&gt; C。线虫</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Research

JoVE Journal

Neuroscience

Automated Quantification of Synaptic Fluorescence in C. elegans

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

含量测定β-淀粉样蛋白的毒性，使用转基因 C。线虫模型

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

科研

神经科学

In vivo Laser Axotomy in C. elegans

In vivo Laser Axotomy in C. elegans

在 C 在体内激光干切断线虫

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

C. elegans Tracking and Behavioral Measurement

C. elegans Tracking and Behavioral Measurement

线虫跟踪和行为测量

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

长期的嗅觉适应性的分子读数<em&gt; C.线虫</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

嵌入后突触蛋白的免疫标记海马切片培养

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging in C. elegans

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

可视化的神经细胞胞质分裂过程中<em&gt; C。线虫</em&gt;胚胎

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

在永久性幼虫特有的神经重构的C语言活体成像线虫

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

的自动分析<em&gt;℃。线虫</em&gt;游泳行为使用天书软件

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...