Name: preparation of mouse brain tissue for immunoelectron microscopy
Uploaded: 2010-07-20T17:45:00
Description: Watch this Scientific Journal Video about Preparation of Mouse Brain Tissue for Immunoelectron Microscopy at JoVE.com

我们感谢少明路和哈里斯答：格尔巴德蠕动泵和vibratome的使用，以及在电子显微镜下研究的罗切斯特大学医学中心为技术援助的核心设施，克伦L. Bentley和盖尔施耐德。这项工作是由来自美国国立卫生研究院（EY019277），白厅基金，宝来惠康基金，和艾尔弗雷德斯​​隆基金会的AKM的资助。 M.-È.T.是由一个全宗RECHERCHE EN桑特魁北克（FRSQ）博士后奖。

1。动物灌注<ol><li>在灌注前一天，准备： - 2 L的磷酸盐缓冲液（PB，100毫米，pH值7.4）和1 L的磷酸钠缓冲液（PBS，0.9％NaCl的50 mM的PB，pH值7.4），双蒸水。存储PB的PBS和1L在4 ° C。这些将被用于灌注和预嵌入免疫细胞化学。 - 1L的4.0％多聚甲醛固定液（煤灰，pH值7.4），这将使灌注6只小鼠。为此，升温1 PB的大号下通风柜。称取40克颗粒多聚甲醛（PFA）的PB液倒入60 ° C，当它到达当该解决方...

<ol>
	<li>Venable, J. H., Coggeshall, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+lead+citrate+stain+for+use+in+electron+microscopy.">A simplified lead citrate stain for use in electron microscopy.</a> J Cell Biol. 25, 407-407 (1965).</li><li>Riad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatodendritic+localization+of+5-HT1A+and+preterminal+axonal+localization+of+5-HT1B+serotonin+receptors+in+adult+rat+brain.">Somatodendritic localization of 5-HT1A and preterminal axonal localization of 5-HT1B serotonin receptors in adult rat brain.</a> J Comp Neurol. 417 (2), 181-181 (2000).</li><li>Tremblay, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+EphA4+in+axon+terminals+and+dendritic+spines+of+adult+rat+hippocampus.">Localization of EphA4 in axon terminals and dendritic spines of adult rat hippocampus.</a> J Comp Neurol. 501 (5), 691-691 (2007).</li><li>Tremblay, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+course+of+EphA4+cellular+and+subcellular+localization+in+the+postnatal+rat+hippocampus.">Developmental course of EphA4 cellular and subcellular localization in the postnatal rat hippocampus.</a> J Comp Neurol. 512 (6), 798-798 (2009).</li><li>Bouvier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-synaptic+and+post-synaptic+localization+of+EphA4+and+EphB2+in+adult+mouse+forebrain.">Pre-synaptic and post-synaptic localization of EphA4 and EphB2 in adult mouse forebrain.</a> J Neurochem. 106 (2), 682-682 (2008).</li><li>Bouvier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EphA4+is+localized+in+clathrin-coated+and+synaptic+vesicles+in+adult+mouse+brain.">EphA4 is localized in clathrin-coated and synaptic vesicles in adult mouse brain.</a> J Neurochem. , (2010).</li></ol>

在这里，我们描述了小鼠脑组织的准备，免疫电镜，最佳的超微结构的保存及免疫细胞化学检测时严格执行程序之间提供了一个很好的折衷协议。 用透射电子显微镜相结合，这种方法可以区分具有几个鲜明的特点和鉴定标准的蜂窝元素。特别是，Iba1免疫过氧化物酶- DAB染色，使确定的小胶质细胞perikarya和流程内的神经纤维，以及分析它们的形态，细胞器超微结构的关系，并?...

*应始终工作在通风橱和磨损丁腈手套时，丙烯醛，多聚甲醛，锇，也应妥善处理其废物。

preparation of mouse brain tissue for immunoelectron microscopy

透射电子显微镜（TEM）是非常有用的可视化的小胶质细胞，oligodendrocytic，星形胶质细胞和神经细胞的亚细胞（树突棘，枝蔓，轴突，轴突终端，perikaryon），以及他们的细胞器和细胞骨架在中枢神经系统，高空间分辨率。预嵌入免疫细胞化学与透射电镜相结合，允许具有几个鲜明的特点和鉴定标准（例如，小胶质细胞perikarya和流程，当使用一种抗体对小胶质细胞特异性标志物Iba1（钙离子结合适配器分子1的细胞成分的歧视;提出在这里）），识别细胞分子神经递质的内容（如五羟色胺）和可溶性或膜结合的蛋白质的超微结构定位（例如，5 HT1A和EphA4受体）。在这里，我们描述了协议transcardiac灌注小鼠丙烯醛固定液，拆除和大脑切片，以及免疫过氧化物酶 - 二氨基联苯胺（DAB）染色，树脂包埋，以及超薄的脑切片切片。这些程序完成后，免疫染色的材料是用透射电子显微镜检查的准备。如果严格执行，这种技术之间最佳的超微结构的保存及免疫细胞化学检测提供了一个很好的折衷。

Watch this Scientific Journal Video about Preparation of Mouse Brain Tissue for Immunoelectron Microscopy at JoVE.com

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

我们描述transcardiac灌注小鼠，拆除和切片的大脑，以及免疫过氧化物酶染色，树脂包埋，和脑切片切片超薄协议。这些程序完成后，免疫染色材料透射电子显微镜检查的准备。

制备小鼠脑组织中的免疫电镜

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

Research

JoVE Journal

Neuroscience

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

聚焦离子束铣削和脑组织的扫描电子显微镜

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

科研

神经科学

Multiphoton Microscopy of Cleared Mouse Brain Expressing YFP

多光子显微镜的清除小鼠脑组织表达YFP

Multiphoton microscopy of intrinsic fluorescence and second harmonic generation (SHG) of whole mouse organs is made possible by optically clearing the ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

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 ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

新生大鼠脑组织在神经端子突形圆锥花序分布超微结构形态分析中的制备

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

鼠脑组织疏水组织清除方法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...