Name: preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient
Uploaded: 2011-09-17T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient at JoVE.com

我们要感谢来自威斯康星大学麦迪逊分校电子显微镜设施电子显微镜的大学BK八月。这项工作是由NIH资助R01 - DA026067和P30 - HD03352（JSM）的支持。

1。准备工作1-4 <ol><li>准备一个2.5米的蔗糖浆混合50毫升，500毫升（GM），梯度介质的缓冲，2.5毫升的一个1M的Tris -盐酸，PH7.5股票，0.1毫升0.5 M EDTA，pH值8.0股毫Q水体积。过滤消毒的解决方案，分装和储存冷冻。 </li><li>准备的河豚毒素（TTX）的1000倍的股票，由1毫米的解决方案中的 Milli - Q H 2 O TTX的等分可以储存在-20 ° C </li><li>准备由Milli - Q水解决了100毫米的Tris（pH值7.5），5毫米?...

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不连续Percoll蔗糖梯度准备此处所描述的是一个快速，可靠的方法来隔离活跃的SNS，它可以用于各种突触传递的实验。此渐变的方法，它是根据Dunkley等人，3,4开发的方法是一种细胞内的脑分馏过程分离前和突触后膜囊泡与另一个相关的衍生。没有进一步纯化这些SNS兼容多种分子生物学技术，包括免疫，免疫印迹，流式细胞仪，监测从头蛋白质的合成，通过[35S] - MET注册成立后，电子?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>微BCA蛋白检测试剂盒</td><td>刺穿</td><td> 23235 </td><td></td></tr><tr><td> 氯化钙 </td><td>费舍尔</td><td> C79 - 500 </td><td></td></tr><tr><td> CO 2气体</td><td>公司Airgas（UW - MDS） </td><td>光盘50 </td><td></td></tr><tr><td> EDTA的</td><td>零售物价指数</td><td> E57020 </td><td></td></tr><tr><td>乙醇</td><td>费舍尔</td><td> A407SK - 4 </td><td></td></tr><tr><td>盐酸</td><td>费舍尔</td><td> A142 - 212 </td><td></td></tr><tr><td> Percoll </td><td> GE医疗集团</td><td> 17-0891-01 </td><td></td></tr><tr><td> KH 2 PO 4 </td><td>费舍尔</td><td> P285 - 500 </td><td></td></tr><tr><td> PierceSDS - PAGE样品制备试剂盒</td><td>刺穿</td><td> 89888 </td><td></td></tr><tr><td>氯化钠</td><td>零售物价指数</td><td> S23020 </td><td></td></tr><tr><td>碳酸氢钠3 </td><td>费舍尔</td><td> BP328 - 500 </td><td></td></tr><tr><td>娜2保护海港条例“4 </td><td>费舍尔</td><td> S381 - 500 </td><td></td></tr><tr><td>蔗糖</td><td>零售物价指数</td><td> S24060 </td><td></td></tr><tr><td> Tris碱</td><td>零售物价指数</td><td> T60040 </td><td></td></tr><tr><td>河豚毒素</td><td>西格玛</td><td> T5651 </td><td></td></tr><tr><td>快临标签混合S35简易标签</td><td> Perkin Elmer公司</td><td> NEG772 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><th>设备</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>解剖工具</td><td></td><td></td><td></td></tr><tr><td> Dounce匀浆，7毫升（带有两个玻璃杵标记为“A”和“B”） </td><td>惠顿</td><td></td><td></td></tr><tr><td> P1000的吉尔森Pipetman </td><td>吉尔森</td><td> F123602 </td><td></td></tr><tr><td> Allegra的6KR离心机</td><td> Beckman Coulter公司</td><td> 366830 </td><td></td></tr><tr><td>生长激素3.8转子，水平转子</td><td> Beckman Coulter公司</td><td> 360581 </td><td></td></tr><tr><td>贝克曼J2 - 21型离心机</td><td>贝克曼</td><td></td><td></td></tr><tr><td>贝克曼管与帽</td><td>贝克曼</td><td> 355672 </td><td></td></tr><tr><td>白色围墙适配器</td><td>贝克曼</td><td> 342327 </td><td></td></tr><tr><td>蓝壁适配器</td><td>贝克曼</td><td></td><td></td></tr><tr><td> JA - 17转子，固定角转子</td><td>贝克曼</td><td> 369691 </td><td></td></tr></tbody></table>用于免疫印迹抗体表： <table border="1"><tbody><tr><th>抗体名称</th><th>公司</th><th>目录编号</th><th>宿主物种</th><th>稀释倍数</th></tr><tr><td> β-肌动蛋白</td><td>西格玛</td><td> A5441 </td><td>小鼠</td><td> 1:2000 </td></tr><tr><td> GFAP </td><td>圣克鲁斯</td><td> SC - 65343 </td><td>小鼠</td><td> 1:200 </td></tr><tr><td> GP73 </td><td>圣克鲁斯</td><td> SC - 134509 </td><td>兔</td><td> 1:200 </td></tr><tr><td> HSC70 </td><td>圣克鲁斯</td><td> SC - 7298 </td><td>小鼠</td><td> 1:200 </td></tr><tr><td> Laminβ </td><td>圣克鲁斯</td><td> SC - 6261 </td><td>山羊</td><td> 1:200 </td></tr><tr><td> Prohibitin </td><td>圣克鲁斯</td><td> SC - 28259 </td><td>兔</td><td> 1:200 </td></tr><tr><td> PSD95 </td><td> Millipore公司</td><td> MAB1596 </td><td>小鼠</td><td> 5微克/μL </td></tr><tr><td> SNAP25 </td><td> Abcam公司</td><td> ab5666 - 100 </td><td>兔</td><td> 1:2000 </td></tr><tr><td>突触</td><td> Millipore公司</td><td> MAB368 </td><td>小鼠</td><td> 1:500 </td></tr><tr><td> β- 3 -微管蛋白</td><td>圣克鲁斯</td><td> SC - 80016 </td><td>小鼠</td><td> 1:200 </td></tr></tbody></table>

preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient

Synaptoneurosomes（SNS）小鼠大脑皮质的同质化和分馏后获得的。他们再封好，水疱或离开时，皮层组织匀浆脱离的轴突终端的隔离终端。 SNS的保留前和突触后的特点，这使得它们在突触传递的研究非常有用。他们保留用于神经信号的分子机制，并能够吸收，储存和释放神经递质。 积极SNS的生产和隔离可能会出现问题的使用聚蔗糖，可细胞毒性，并要求延长离心由于密度高，过滤和离心的方法，它可以在低活动导致机械损伤的SNS等媒体。然而，使用不连续Percoll蔗糖密度梯度分离SNS提供了一种快速的方法来产生良好收益的翻译活跃的SNS。 Percoll蔗糖梯度法快速，温和的，因为它采用等渗条件下，有越来越短的离心旋转，并避免离心步骤，颗粒SNS和造成机械损伤。

Watch this Scientific Journal Video about Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient at JoVE.com

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

一个准备从小鼠大脑皮质翻译活跃，完好synaptoneurosomes（SNS）的方法来描述。该方法采用一个不连续Percoll蔗糖密度梯度，允许快速制备活跃的SNS。

准备使用一个不连续Percoll蔗糖密度梯度的从小鼠皮质Synaptoneurosomes

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

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Neuroscience

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

慢性成像小鼠的视觉皮层使用减薄，颅骨的制备

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

科研

神经科学

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

关联从人类前额叶皮层的fMRI信号的行为反应：检查的认知过程，使用任务分析

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

隔离LacZ基因表达的小鼠内耳组织用流式细胞仪检测细胞

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

听觉皮层在成年猫用高场磁共振成像功能成像

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

突触质膜和突触后密度蛋白采用不连续蔗糖梯度准备

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex

调查深皮层的功​​能和皮层下结构采用立体定向脑电图：由前扣带皮层的经验教训

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure ...