Name: correlative light and electron microscopy to study microglial interactions with &#946;-amyloid plaques
Uploaded: 2016-06-01T15:00:00
Description: Watch this Scientific Journal Video about Correlative Light and Electron Microscopy to Study Microglial Interactions with ÃŽÂ²-Amyloid Plaques at JoVE.com

We are grateful to Dr. Sachiko Sato and Julie-Christine L&#233;vesque at the Bioimaging Platform of the Centre de recherche du CHU de Qu&#233;bec for their technical assistance. Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN-2014-05308, The Banting Research Foundation, and The Scottish Rite Charitable Foundation of Canada to M.E.T supported this work.
H.E.H. is recipient of a scholarship from the Lebanese Ministry of Education and Higher Education, and K.B. from the Facult&#233; de m&#233;decine of Universit&#233; Laval.

注：所有实验均批准并根据机构的动物伦理委员会的指导方针与加拿大议会关于动物饲养的准则进行的，符合由拉瓦尔大学的动物护理委员会的管理。使用了4至21个月的年龄之间的APP-PS1雄性小鼠。 25℃，免费获得食物和水 - 这些动物在22一12小时明暗周期下安置。 1.甲氧基X04溶液制备<ol><li>由甲氧基X04溶解于含有10％二甲基亚砜（DMSO），45％丙二醇和45％磷酸钠缓冲盐水（在1...

<ol>
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该协议解释了使用EM针对致密核心Aβ斑块相关办法。甲氧基X04 体内注入允许包含特定区域内的Aβ斑块和感兴趣的层，例如海马CA1区，地层radiatum和腔隙-moleculare脑切片的快速选择。在本例中，甲氧基X04预检用嵌入预免疫染色IBA1研究小神经胶质表型如何不同与在致密核心的Aβ斑块的存在超微结构水平突触相互作用结合。 小胶质细胞是令人难以置信的响应他们的环境和他们...

淀粉样蛋白的Aβ斑块的形成是阿尔茨海默氏病（AD）的主要神经病理学特征。然而越来越多的证据表明，在疾病进展1,2-免疫系统的重要的作用。特别是，从临床前和临床研究的新的数据建立的免疫功能障碍为主要驱动和贡献者AD病理学。有了这些发现，中枢及外周免疫细胞已成为有前途的治疗靶点AD 3。以下方案结合了光学和电子显微镜（EM），以产生新的见解的Aβ斑块沉积和在公元小胶质细胞表型改变之间的关系。该协议允许的Aβ斑块在AD小鼠模型中使用的荧光染料甲氧基X04的体内注射的标记。甲氧基X04是，可以很容易穿越血 - 脑屏障进入脑实质并结合以高的β型褶片材的刚果红衍生物ffinity。因为染料是荧光的，它可用于Aβ斑块沉积的体内检测用双光子显微镜4。一旦绑定到Aβ，甲氧基X04不会离解或斑块重新分配了，而且随着时间的推移保留其荧光。它一般给药外周，以允许大脑动力学5的非侵入性成像。荧光也保持以下醛固定，允许相关验尸分析，包括在Aβ斑块6附近的神经元死亡的调查。 这个协议利用甲氧基X04的性质来选择由APP SWE / PS1A 246E小鼠脑切片（APP-PS1;共表达在APP基因Lys670Asn / Met671Leu一个双突变，和人类早老PS1-A264E变体）表现出Aβ7感兴趣的特定区域（海马CA1，地层radiatum和腔隙，moleculare斑）预嵌入对小胶质细胞标记钙离子结合适配器分子1（IBA1免疫）可视化小胶质细胞机构和EM进程之前。将小鼠通过穿心灌注给定的甲氧基X04溶液，脑定影前24小时腹膜内注射。使用振动得到冠状脑切片。含有海马CA1节为Aβ斑块在地层radiatum和腔隙-moleculare存在下在荧光显微镜下进行筛选。免疫染色IBA1，四氧化锇后固定，塑料树脂包埋在所选脑切片随后进行。在该协议的结束时，部分可以在没有进一步的超微结构降解被存档，准备超薄切片和超微结构检查。重要的是，斑块仍与不同的抗体的免疫染色，例如IBA1如在本协议之后荧光。他们变得更暗Ť汉其周围神经毡以下锇酸后固定，独立地甲氧基X04染色，这有助于准确地识别感兴趣的区域，一般下降到几平方毫米，以用透射电子显微镜进行检查。 这种关联方法提供了一个有效的方式来确定具体的脑切片在超微结构层面来考察。研究早期AD病理学，特定脑区域或层内，可能只包含几个Aβ斑块中，存在于只有组织切片的一小部分时，这是特别有帮助。在这些时候尤其，这将是低效率的用于Aβ免疫染色（和双标记用于其它细胞标记诸如IBA1）在几个脑切片简单地，得到含有的Aβ斑块在合适位置的一小部分。此外，注射活体小鼠与甲氧基X04牺牲和组织处理没有做之前，仲裁协议Ë超微结构保存。替代方法，如刚果红，硫磺素S，硫黄素T或甲氧基X04验染色固定组织切片需要乙醇染色分化，8-11导致渗透压和破坏的超微结构。刚果红也是已知的人类致癌物质12。

<table border="0" cellpadding="0" cellspacing="0" width="1006">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Methoxy-X04</td>
			<td style="width:197px;">Tocris Bioscience</td>
			<td style="width:119px;">4920</td>
			<td style="width:167px;">10 mg substrate per tablet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propylene glycol</td>
			<td>Sigma Aldrich</td>
			<td>W294004&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher BioReagents</td>
			<td>BP231-1</td>
			<td>Caution: toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride NaCl</td>
			<td>Sigma</td>
			<td>S9625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate monobasic monohydrate</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S0876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Hydrochloride</td>
			<td>Fisher BioReagents</td>
			<td>BP153500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acrolein&#160;</td>
			<td>Sigma</td>
			<td>110221</td>
			<td>Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde Granular</td>
			<td>Electron Microscopy Sciences</td>
			<td>19210</td>
			<td>Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter paper</td>
			<td>Fisher</td>
			<td>09-790-14F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peristaltic Pump with Tubing</td>
			<td>ColeParmer</td>
			<td>cp.78023-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Excel Winged blood Collection Set Needles 25G&#160;</td>
			<td>Becton Dickinson</td>
			<td>367341</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Extrafine Forceps</td>
			<td>F.S.T</td>
			<td>11152-10</td>
			<td>Tip shape: curved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors</td>
			<td>F.S.T</td>
			<td>14090-09</td>
			<td>Tip shape: straight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hartman Hemostats</td>
			<td>F.S.T</td>
			<td>13003-10</td>
			<td>Tip shape: curved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical Scissors</td>
			<td>F.S.T</td>
			<td>14004-16</td>
			<td>Tip shape: straight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Dissecting Scissors</td>
			<td>ROBOZ surgical store</td>
			<td>5818</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>74515-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibrating Blade Michrotome Leica VT1000 S</td>
			<td>Leica Biosystems&#160;</td>
			<td>14047235612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome blades</td>
			<td>Electron microscopy Sciences</td>
			<td>71990</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Tissue Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylene Glycol</td>
			<td>Fisher BioReagents</td>
			<td>BP230-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>BP229-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrogen Peroxide, 30%</td>
			<td>J.T.BAKER</td>
			<td>cat: 2186-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium borohydride</td>
			<td>Sigma</td>
			<td>480886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris HCl</td>
			<td>Fisher</td>
			<td>BP153-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine Serum (FBS)</td>
			<td>Sigma Aldrich</td>
			<td>F1051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA), fraction V</td>
			<td>Thomas Scientific</td>
			<td>C001H24</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti IBA1, Rabbit&#160;</td>
			<td>WAKO</td>
			<td>019-19741</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rabbit IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>111066046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASTAIN Elite ABC Kit (Standard)</td>
			<td>Vector Labs</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3.3&#39;-Diaminobenzidine tetra-hydrochloride (DAB)</td>
			<td>Sigma</td>
			<td>D5905-50TAB</td>
			<td>Caution: toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Osmium tetroxide, 4% solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>19150</td>
			<td>Caution: toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component A</td>
			<td>Sigma</td>
			<td>44611</td>
			<td>Resin Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component B</td>
			<td>Sigma</td>
			<td>44612</td>
			<td>Hardener Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component C</td>
			<td>Sigma</td>
			<td>44613</td>
			<td>Plasticizer Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component D</td>
			<td>Sigma</td>
			<td>44614</td>
			<td>Accelerator Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>LesAlcoolsdeComerce</td>
			<td>151-01-15N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propylene oxide</td>
			<td>Sigma</td>
			<td>110205</td>
			<td>Caution: corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum weigh dishes</td>
			<td>Electron Microscopy Sciences</td>
			<td>70048-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACLAR&#174;&#8211;Fluoropolymer Films</td>
			<td>Electron Microscopy Sciences</td>
			<td>50425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oven/Incubator</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

correlative light and electron microscopy to study microglial interactions with &#946;-amyloid plaques

详细协议这里提供给前从阿尔茨海默氏病小鼠模型中确定在脑切片的淀粉样蛋白的Aβ斑块的预嵌入免疫染色（特别为离子化的钙结合衔接分子​​1（IBA1），由小胶质细胞中表达的钙结合蛋白）和组织处理电子显微镜（EM）。甲氧基X04是荧光染料跨越血 - 脑屏障和选择性结合在致密核心的Aβ斑块中发现β-打褶纸。用甲氧基X04动物注射前牺牲和脑定影允许预筛分和含斑块的脑切片进行进一步的处理与费时的操作的选择。研究可能含有非常少的斑块，本仅在部分的一小部分的特定的脑区域或层内早期AD病理时，这是特别有帮助。刚果红，硫磺素S和Thiofl组织切片的验尸处理AVIN T（或者甚至与甲氧基X04）可以标记β-折叠片，但需要用乙醇广泛清算去除多余的染料和这些程序与超微结构保存不兼容。它也将是低效率的，以对Aβ（和其它细胞标记诸如IBA1）对感兴趣的区域的所有的脑切片进行标记，只得到含有的Aβ斑块在合适位置的一小部分。重要的是，Aβ斑块仍然可见的EM组织处理后，允许对区域的精确识别（一般下降到几平方毫米），用电子显微镜检查。

此部分示出了可以在协议的不同的关键步骤得到的结果。特别是，结果显示含有在特定区域甲氧基X04染色斑块和感兴趣的层的脑切片的例子：海马CA1区，地层radiatum和腔隙-moleculare。的斑块和海马区域/层状组织使用UV和亮场滤波器（ 图1）的组合依次显示。所选的脑切片随后免疫染色并处理的EM同时跟踪其的Aβ斑块的，考虑到它们仍然荧光以下免疫染色，并在与...

Watch this Scientific Journal Video about Correlative Light and Electron Microscopy to Study Microglial Interactions with ÃŽÂ²-Amyloid Plaques at JoVE.com

Correlative Light and Electron Microscopy to Study Microglial Interactions with ÃƒÅ½Ã‚Â²-Amyloid Plaques

本文介绍了使用甲氧基X04，它穿过血 - 脑屏障，并选择性地结合β褶的致密核心斑块Aβ发现张在阿尔茨海默氏症小鼠模型可视化淀粉样蛋白斑块Aβ的协议。它允许的前免疫染色和处理电子显微镜含斑块组织切片预检。

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Research

JoVE Journal

Neuroscience

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

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

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

光偏好的检测，以研究的先天和昼夜调节感光特性<em&gt;果蝇</em&gt;幼虫

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

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 ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

研究病原体或药物交叉障碍及其与大脑相互作用的人血脑界面模型

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

使用 PRIMO 微模式系统对蛋白质的光诱导分子吸附，研究细胞对细胞外基质蛋白的反应

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

从鸡胚胎的直膜到研究棒到锥细胞相互作用的锥形丰富文化

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

Serial Block-Face Scanning Electron Microscopy (SBEM) for the Study of Dendritic Spines

Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines

用于登德里脊柱研究的串行块面扫描电子显微镜 （SBEM）

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

大鼠星形胶质细胞和小胶质细胞的原代培养及其在肌萎缩侧索硬化症研究中的应用

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

相关光学和电子显微镜，研究与β淀粉样蛋白斑块的小神经胶质互动