Name: wholemount immunohistochemistry for revealing complex brain topography
Uploaded: 2012-04-05T13:00:00
Description: Watch this Scientific Journal Video about Wholemount Immunohistochemistry for Revealing Complex Brain Topography at JoVE.com

室间隔新Yeshiva大学爱因斯坦医学院的研究者从启动资金支持。

1。动物灌注和小脑剖析<ol><li>根据蛋白质，灌注可能是必不可少的成功染色1,2。 transcardiac灌注是一种侵入性，非生存的过程，需要正确使用的麻醉药。正确的培训，机构批准，并IACUC批准，都在尝试的过程之前，必要的。它始终是一个好主意，咨询机构的兽医，以确定实验的要求，并获得正确的训练中得到的帮助。手术前，动物应该是深度麻醉，没有反应，如脚或尾巴捏刺激。用剪刀?...

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我们已经介绍了使用一个多功能的免疫组织化学方法，在发展中国家和成人的大脑暴露蛋白表达为成功wholemount的染色所需的技术细节。复杂的分子的表达方式，通过使用这种方法，可以分析和脑地形而不需要费时费力组织切片程序赞赏。 该协议已被用于揭示成人1,2,8,9和产后早期小鼠小脑10,11,12图案几个Purkinje细胞蛋白表达。我们原来的研究也表明了实用的的?...

<table border="1"><tbody><tr><td> 物料 </td><td> 在协议中的作用 </td></tr><tr><td>灌注泵（费舍尔Scientific/13-876-2） </td><td>允许一致的和缓慢灌注。 </td></tr><tr><td>夏普尖端剪刀（FST/14081-08） </td><td>一般用在灌注和解剖。 </td></tr><tr><td>钝尖钳（FST/91100-12） </td><td>为了稳定灌注针插入心脏。 </td></tr><tr><td>镊子（杜蒙AA/11210-10由福斯特） </td><td>对于使用过程中的脑由颅骨的解剖和大脑的其余部分分开小脑。这些都是必要的，因为他们有一个尖端略圆，有助于最大限度地减少损害小脑在解剖。 </td></tr><tr><td> nutator（Fisher Scientific则） </td><td>用来保持在运动过程中的潜伏期组织。 </td></tr><tr><td> 1.5 mL管（萨尔斯塔特/螺帽微管） </td><td>在他的所有步骤组化协议，在这些微管的地方。圆润的底部，确保小脑在运动中保持。 </td></tr><tr><td>穿孔勺（FST/10370-17） </td><td>用于轻轻滗出花的解决方案保持在微管wholemounts。 </td></tr><tr><td>徕卡MZ16足总杯显微镜</td><td>用于检查wholemount染色。 </td></tr><tr><td>的徕卡DFC3000 FX相机</td><td>用来捕捉wholemount图像。 </td></tr></tbody></table> 表1。 <table border="1"><tbody><tr><td colspan="9"> 例如日历典型wholemount实验 </td></tr><tr><td>第1天</td><td colspan="4">登特的修复，室温，8小时</td><td colspan="4">登特的漂白剂，4℃，过夜</td></tr><tr><td>第2天</td><td colspan="3"> 100％甲醇，室温，2X，每次30分钟</td><td colspan="3"> 100％甲醇，冻结/解冻， 4倍，30 min/15分钟</td><td colspa<html> N =“2”&gt; 100％甲醇，80°，隔夜</td></tr><tr><td>第3天</td><td> 50％MeOH/50％PBS，室温60-90分钟</td><td colspan="2"> 15％甲醇/ 85％PBS，室温60-90分钟</td><td colspan="2"> 100％PBS，室内温度60-90分钟</td><td> 10μg/mL组蛋白酶K在PBS，室温2-3分钟</td><td> 100％FBS，室温，3倍，每10分钟</td><td>光电倍增管，4℃，过夜</td></tr><tr><td> 4-5天</td><td colspan="8">光电倍增管+ 1°抗体+ 5％DMSO，4°C间，48小时</td></tr><tr><td>第6天</td><td colspan="3">光电倍增管，4℃，2 - 3倍，每2-3小时</td><td colspan="5">光电倍增管+ 2°抗体+ 5％DMSO，4°C间，24小时（或开始与ABC复杂扩增步骤） </td></tr><tr><td> 7天</td><td colspan="2">光电倍增管，4℃，2 - 3倍，每2-3小时</td><td colspan="3"> PBT，室温，2小时; </td><td colspan="3">新鲜民建联在PBS孵育，直到最佳染色是可视化</td></tr></tbody></table>_content“&gt; 表2。 <table border="1"><tbody><tr><td colspan="2"> 食谱 （* =每次都准备好新鲜） </td></tr><tr><td> PBS（磷酸盐缓冲液） </td><td> 0.1M磷酸盐缓冲液中去离子水。 pH值7.2（西格玛片; P4417） </td></tr><tr><td> PFA（聚甲醛） </td><td>制造和储存冻结20％的解决方案，然后4％的PBS稀释为工作液（Fisher Scientific则; T353） </td></tr><tr><td>登特的定影液3 * </td><td> 4部分甲醇 第1部分二甲基亚砜（二甲基亚砜; Fisher Scientific则; D159-4） </td></tr><tr><td>登特的漂白剂3 * </td><td> 4部分甲醇 第1部分二甲基亚砜（二甲基亚砜; Fisher Scientific则; D159-4） 第1部分30％的过氧化氢</td></tr><tr><td>酶解</td><td> 10微克/毫升的PBS，蛋白酶K（罗氏诊断; 03115828001）。 </td></tr><tr><td> PBST </td><td> PBS包含： 0.1％的吐温20（Fisheŕ科学，BP337;海卫也可以在所有情况下使用Tween-20的地方）。 </td></tr><tr><td>光电倍增管25 * </td><td> PBS包含： 2％脱脂脱脂奶粉（康乃馨优先） 0.1％Tween-20的（Fisher Scientific则BP337） </td></tr><tr><td> PBT的25 * </td><td> PBS包含： 0.2％的牛血清白蛋白（Sigma公司; B9001S） 0.1％Tween-20的（Fisher Scientific则BP337） </td></tr><tr><td>民建联* </td><td>溶解在40毫升的PBS 3,3 - 二氨基联苯胺10毫克片剂（Sigma-Aldrich公司; D5905）。加入10μl30％的过氧化氢的启动反应）。 </td></tr><tr><td> ABC复杂的解决方案</td><td> vectastain套件（媒介实验室，公司PK-4000） </td></tr></tbody></table> 表3。

wholemount immunohistochemistry for revealing complex brain topography

反复和充分了解小脑的细胞结构，使其理想的模型系统，为探索脑地形。其相对统一的细胞结构的基础是一系列复杂的基因和蛋白表达的矢状域。小脑分子条块分割镜像传入纤维的解剖和功能的组织。要充分认识小脑组织的复杂性，我们以前完善了wholemount染色方法在小鼠小脑的图案缺陷的高通量分析。该协议详细描述的试剂，工具，是有用的实际步骤，成功地揭示了成年小鼠小脑使用wholemount染色蛋白表达模式。这里展示的步骤强调使用这种方法的效用作为一个例子，如何可以在其透露的大脑的精细地形表达zebrinII / aldolaseC的原生立体构象。还介绍了协议允许传入的预测和大量分子地形比较研究小脑蛋白表达的可视化的适应。为了说明这些应用程序，从大鼠小脑的传入染色的数据。

Watch this Scientific Journal Video about Wholemount Immunohistochemistry for Revealing Complex Brain Topography at JoVE.com

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

神经回路地形组织功能的车厢与特定的分子型材。在这里，我们提供一个多功能wholemount免疫组织化学染色方法为揭示全球脑地形使用的实际和技术步骤。我们证明了实用的使用方法很好理解的小脑细胞构筑和电路。

为揭示复杂的大脑地形wholemount免疫组化

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

Research

JoVE Journal

Neuroscience

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

脑室下区恩的脸：Wholemount染色和室管膜流量

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

科研

神经科学

Revealing Neural Circuit Topography in Multi-Color

多色揭示神经回路地形

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

在Wholemount大鼠视网膜免疫组化和钙成像方法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

引物对免疫冰冻切片大鼠脑组织：例为染色小胶质细胞和神经元

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

使用电动和一维和二维文化磁场神经元的外部激励

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

非人类灵长类动物脑组织的制备预嵌入免疫组化和电子显微镜

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

常见马莫塞的全皮质电皮质阵列的慢性植入

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Purification of the Dendritic Filopodia-rich Fraction

富德瑞迪蒂菲洛波迪亚成分的纯化

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

小鼠视网膜冷冻节的制备,用于免疫性植物化学

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...