Name: using near-infrared fluorescence and high-resolution scanning to measure protein expression in the rodent brain
Uploaded: 2019-05-23T15:00:00
Description: Watch this Scientific Journal Video about Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain at JoVE.com

本报告中的研究由 NIGMS 的目标赠款 (1P20GM103653) 资助, 授予 DK。

以下议定书得到了特拉华州大学动物护理和使用机构委员会 (IACUC) 的批准。大约55-75天大的雄性 Sprague Dawley 大鼠被用于该方案。
1. 脑摘除和组织准备
<ol><li>将大鼠在麻醉诱导室中使用异氟烷, 直到大鼠不再对脚夹紧有反应。</li>
	<li>用断头台通过快速斩首来牺牲老鼠。</li>
	<li>将头骨的皮肤切割到前部, 从头骨顶部清除。</li>
	<li>使用公语仔细地取出头骨的后部, 然后使用小解...

<ol>
	<li>Kandel, E. 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> Principles of Neural Science. , (2013).</li><li>Byrne, J. H., Roberts, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> From Molecules to Networks: An Introduction to Cellular and Molecular Neuroscience. , (2009).</li><li>Salami, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+D2/3+binding+potential+modulates+neural+signatures+of+working+memory+in+a+load-dependent+fashion.">Dopamine D2/3 binding potential modulates neural signatures of working memory in a load-dependent fashion.</a> Journal of Neuroscience. , (2018).</li><li>Merali, Z., Khan, S., Michaud, D. S., Shippy, S. A., Anisman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+amygdaloid+corticotropin-releasing+hormone+(CRH)+mediate+anxiety-like+behaviors?+Dissociation+of+anxiogenic+effects+and+CRH+release.">Does amygdaloid corticotropin-releasing hormone (CRH) mediate anxiety-like behaviors? Dissociation of anxiogenic effects and CRH release.</a> European Journal of Neuroscience. 20 (1), 229-239 (2004).</li><li>English, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dataset+of+mouse+hippocampus+profiled+by+LC-MS/MS+for+label-free+quantitation.">Dataset of mouse hippocampus profiled by LC-MS/MS for label-free quantitation.</a> Data in Brief. 7, 341-343 (2016).</li><li>David, M. A., Tayebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+protein+aggregates+in+brain+and+cerebrospinal+fluid+derived+from+multiple+sclerosis+patients.">Detection of protein aggregates in brain and cerebrospinal fluid derived from multiple sclerosis patients.</a> Frontiers in Neurology. 5, 251 (2014).</li><li>Oliver, C., Jamur, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+methods+and+protocols.">Immunocytochemical methods and protocols.</a> Methods in Molecular Biology. , (2010).</li><li>Spitzer, N., Sammons, G. S., Price, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autofluorescent+cells+in+rat+brain+can+be+convincing+impostors+in+green+fluorescent+reporter+studies.">Autofluorescent cells in rat brain can be convincing impostors in green fluorescent reporter studies.</a> Journal of Neuroscience Methods. 197 (1), 48-55 (2011).</li><li>Knox, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+c-Jun+to+identify+fear+extinction+learning-specific+patterns+of+neural+activity+that+are+affected+by+single+prolonged+stress.">Using c-Jun to identify fear extinction learning-specific patterns of neural activity that are affected by single prolonged stress.</a> Behavioural Brain Researach. 341, 189-197 (2018).</li><li>Knox, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuits+via+which+single+prolonged+stress+exposure+leads+to+fear+extinction+retention+deficits.">Neural circuits via which single prolonged stress exposure leads to fear extinction retention deficits.</a> Learning &amp; Memory. 23 (12), 689-698 (2016).</li><li>Malinow, R., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+trafficking+and+synaptic+plasticity.">AMPA receptor trafficking and synaptic plasticity.</a> Annual Review of Neuroscience. 25, 103-126 (2002).</li><li>Nabavi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+memory+with+LTD+and+LTP.">Engineering a memory with LTD and LTP.</a> Nature. 511 (7509), 348-352 (2014).</li><li>Kimmelmann-Shultz, B., Mohammadmirzaei, N., Della Valle, R., Knox, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+high+resolution+near+infrared+imaging+to+measure+fear-learning+induced+changes+in+AMPA/NMDA+ratios+throughout+the+fear+circuit.">Using high resolution near infrared imaging to measure fear-learning induced changes in AMPA/NMDA ratios throughout the fear circuit.</a> , (2018).</li><li>Eagle, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+prolonged+stress+enhances+hippocampal+glucocorticoid+receptor+and+phosphorylated+protein+kinase+B+levels.">Single prolonged stress enhances hippocampal glucocorticoid receptor and phosphorylated protein kinase B levels.</a> Neuroscience Research. 75 (2), 130-137 (2013).</li></ol>

本文的研究结果表明, 近红外免疫细胞化学结合高分辨率扫描可用于获得脑组织中蛋白质表达的半定量指标。它还可以用来在同一大脑区域同时标记两种蛋白质。我们以前用近红外免疫组织化学来测量多个大脑区域9、10 的直接早期基因表达。即时早期基因可以用来衡量神经活动。我们还将这些蛋白质表达的半定量指标进行了统计分析, 使我们能够对大脑...

神经科学的研究涉及对大脑中细胞如何介导特定功能的研究 1。这些可能是细胞性质的, 例如胶质细胞如何在中枢神经系统中赋予免疫, 或者可能涉及旨在解释海马背侧神经元的活动如何导致空间导航的实验。从广义上讲, 细胞功能是由细胞中表达的蛋白质和这些蛋白质的活性2决定的。因此, 测量脑细胞中蛋白质的表达和活性对于神经科学的研究至关重要。
有许多技术可以用来测量大脑中的蛋白质表达。这些方法包括体内方法, 如受体密度为 3的正电子发射地形和小肽4的微透析。更常见的是, 体外方法被用来检查蛋白质的功能和表达。其中包括质谱技术5、西方印迹和酶联免疫吸附法 (elisa)6和免疫细胞化学7。免疫细胞化学在神经科学领域得到了广泛的应用。这项技术涉及使用初级抗体检测感兴趣的蛋白质 (或抗原) (例如 c-Fos) 和共轭二级抗体来检测蛋白质-初级抗体复合物 (图 1)。为了能够检测蛋白-初级抗体-二级抗体复合物, 二级抗体具有氧化剂, 如辣根过氧化物酶 (HRP) 结合到它们。这样就可以在细胞中形成沉淀物, 可以使用光学显微镜7检测到。继发性抗体也可以使化学物质与之结合在一起 (即荧光)。当受到刺激时, 这些化学物质会发光, 可用于检测蛋白质原代抗体-二级抗体复合物 7。最后, 有时初级抗体具有还原剂和荧光化学物质, 直接否定了对二级抗体的需求 ( 图 1)。
有趣的是, 许多免疫细胞化学方法允许对脑细胞中的蛋白质进行可视化, 但不能量化特定细胞或大脑区域中的蛋白质含量。使用光显微镜检测减少反应的沉淀物可以显示神经元和胶质细胞, 但这种方法不能用于量化蛋白质在细胞或特定大脑区域的表达。从理论上讲, 荧光显微镜可以用于这一点, 因为荧光二级抗体发出的光是对蛋白质-初级抗体-二级抗体复合物的测量。然而, 脑组织中的自体荧光可能会使使用荧光显微镜来量化脑组织中的蛋白质表达。因此, 脑组织荧光图像发出的光很少被用来量化大脑中的蛋白质表达。
其中许多问题可以通过近红外免疫细胞化学与高分辨率扫描9,10一起解决。在本文中, 我们描述了如何免疫细胞化学结合近红外发射光谱中的荧光, 可以结合高分辨率扫描 (例如, 10–21μm), 以获得清晰的图像, 允许在不同的蛋白质半定量大脑区域。

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	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:658px;">Brain Extraction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Anesthesia Induction Chamber</td>
			<td style="width:147px;">Kent Scientific</td>
			<td>VetFlo-0530SM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kleine Guillotine</td>
			<td style="width:147px;">Harvard Apparatus</td>
			<td>73-1920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Friedman Rongeur</td>
			<td style="width:147px;">Fine Science Tools</td>
			<td>16000-14</td>
			<td>used to remove back of skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Delicate Dissecting Scissors</td>
			<td style="width:147px;">Fischer Scientific</td>
			<td>08-951-5</td>
			<td>used to cut upward along midline of skull</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Micro Spatula</td>
			<td style="width:147px;">Fischer Scientific</td>
			<td>21-401-5</td>
			<td>used to scoop out brain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Glass Microscope Slides</td>
			<td style="width:147px;">Fischer Scientific</td>
			<td style="width:114px;">12-549-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:658px;">Immunohistochemical Reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">&#160;Triton X-100</td>
			<td style="width:147px;"></td>
			<td style="width:114px;"></td>
			<td>Used as a mild detergent to permeabilize cells after fixing in Paraformaldehyde, also used as mild detergent in combination with host serum and secondary antibody&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Tween-20</td>
			<td style="width:147px;"></td>
			<td style="width:114px;"></td>
			<td>Used as a small amount of detergent added to TBS&#160; to procuce TBS-T after coverslipping slides with primary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Licor Odyssey scanner</td>
			<td style="width:147px;">Licor Biotechnology Inc.</td>
			<td style="width:114px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Image Studio</td>
			<td style="width:147px;">Licor Biotechnology Inc.</td>
			<td style="width:114px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

using near-infrared fluorescence and high-resolution scanning to measure protein expression in the rodent brain

神经科学是研究大脑中的细胞如何调节各种功能的研究。测量神经元和胶质细胞中的蛋白质表达对于神经科学的研究至关重要, 因为细胞功能是由细胞蛋白的组成和活性决定的。在本文中, 我们描述了免疫细胞化学如何可以结合近红外高分辨率扫描, 以提供在不同的大脑区域的蛋白质表达的半定量测量。这项技术可用于同一大脑区域的单蛋白或双蛋白表达。用这种方式测量蛋白质可以通过实验操作、学习和记忆的分子特征、分子途径中的活动以及多个大脑区域的神经活动来获得蛋白质表达的相对变化。通过正确的蛋白质和统计分析, 也可以确定大脑区域之间的功能连接。鉴于在实验室实施免疫细胞化学很容易, 使用具有近红外高分辨率扫描的免疫细胞化学可以扩大神经学家在系统层面检查神经生物学过程的能力。

在使用高分辨率扫描进行免疫组织化学之前, 应验证该协议是否有效。这可以通过验证分析来实现, 在这种方法中, 来自同一动物的大脑部分用原生和二级抗体、二级抗体单独培养, 或者既不存在原发抗体, 也不使用二级抗体。这种验证分析的结果如图 2所示。在这个反应中, 我们检测到了海马背侧和杏仁核的直接早期基因 c-Jun。只有当一次和二级抗体?...

Watch this Scientific Journal Video about Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain at JoVE.com

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

在这里, 我们提出了一个协议, 使用近红外染料结合免疫组织化学和高分辨率扫描来检测大脑区域的蛋白质。

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

该协议是重要的，因为它允许在同一脑区域的两个蛋白质的定量。这使得实验者能够通过检测泛蛋白和磷蛋白来研究大脑区域分子信号通路的活动。它也可以用来检测蛋白质，这是不同认知现象的标记。我们使用一种相对简单的技术，如免疫组织化学，来回答通常需要更多参与技术的问题。包括检查分子信号通路的活动，检查AMPA/NMDA比率。使用低温恒温保持在零下9摄氏度和零下12摄氏度之间，切片以前分离和冷冻的老鼠大脑在感兴趣的区域在30-50微米。直接将切片安装到玻璃幻灯片上，并在零下 80 摄氏度下储存，直到免疫细胞化学。从冰柜中取出玻璃滑梯，让其平衡到室温 30 分钟。在烟罩下工作，将幻灯片放在 0.1 摩尔 PBS 中的 4% 半甲醛中，在室温下放置一到两个小时，用于组织固定。然后用 0.1 摩尔 TBS 冲洗幻灯片三次，每次 10 分钟。要渗透细胞膜，在温和的洗涤剂中孵育幻灯片30-60分钟。在 TBS 中再次冲洗三次，每次冲洗 15 分钟。稀释PBS中感兴趣的蛋白质的原抗体，浓度正确，如手稿所述。移液原抗体溶液直接到脑组织上。将盖板滑在幻灯片上，在室温下在原抗体稀释中孵育脑组织1-2小时。孵育后，取出盖板，在TBS中清洗含有少量洗涤剂的幻灯片，每次四次，每次15分钟。在含有TBS、洗涤剂和1.5%宿主血清的稀释剂中稀释二次抗体。在幻灯片中加入二次抗体，盖上盖板，在室温下孵育两小时。孵育后，用TBS-T冲洗幻灯片四次，每次20分钟，然后用TBS冲洗四次，每次冲洗20分钟。在黑暗中，在夜间在室温下干燥滑梯。将幻灯片放置到近红外扫描接口上，组织朝下。使用选择工具一次成像多个幻灯片。使用分辨率为 21 微米的最高质量设置对幻灯片进行图像图像。在零纳米偏移中，将图像导入图像分析软件，用于半量化蛋白质分析的查看和标记。打开图像分析软件后，选择扫描图像的工作区。然后在图像分析软件中打开扫描图像，查看扫描并调整显示的波长、对比度、亮度和放大倍率，而不更改原始图像或总量化发射。确定要量化的关键区域后，沿页面顶部选择分析选项卡，然后选择绘制矩形以在要量化的区域上绘制矩形。要查看矩形大小，请选择屏幕左下角的形状。然后沿右下角选择列。然后添加高度和宽度列以标识形状大小。最后，命名形状并重复。采样所有区域后，继续合并和分析列选项卡中可用的数据。为了验证此协议是否有效，大脑部分被孵育为初级抗体和二级抗体，单独使用二级抗体，或既不是初级抗体，也不是二级抗体。只有在对脑组织应用初级抗体和二级抗体时，才能在后海马和杏仁核中检测到直接的早期基因c-jun表达。在杏仁核的粪便蛋白检测中，测定了AMA受体的GluR1亚单元和NMDA受体的NR2A亚单元。这允许检查AMBA NMDA受体在杏仁核亚核的比例，这是学习和记忆的神经生物学特征。蛋白质表达的均值和规范化测量值来自心海马体的高分辨率扫描。使用图像分析软件，在感兴趣的区域放置一个矩形，并且使用形状中平均光强作为荧光表达的度量，这是蛋白质表达的度量。为了获得规范化曲线，在曲线下区域表示高信号和低信号的区域放置了形状，作为蛋白质表达的度量。与这个程序最大的困难是找到一个抗体，并确保它的工作原理。应用程序很简单。我的建议是先尝试一个常规的免疫石化学程序，然后尝试这种技术。始终先执行验证检测。要记住的最重要的事情是检查你的抗体稀释，并确保它正确应用。如果没有这些步骤，该过程将失败。

Research

JoVE Journal

Neuroscience

Simultaneous fMRI and Electrophysiology in the Rodent Brain

同时在啮齿动物脑功能磁共振成像和电

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

科研

神经科学

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

使用近红外光谱MazeSuite和功能研究学习空间导航

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

大脑切片生物素化：一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Personalized Needles for Microinjections in the Rodent Brain

啮齿动物脑 Microinjections 的个性化针

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy (NIRS) Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy NIRS Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa

利用功能近红外光谱成像技术研究全球儿童发展: 农村撒哈拉以南非洲地区的神经影像场方法

Portable neuroimaging approaches provide new advances to the study of brain function and brain development with previously inaccessible populations and in ...