Name: a hydrophobic tissue clearing method for rat brain tissue
Uploaded: 2020-12-23T15:00:00
Description: Watch this Scientific Journal Video about A Hydrophobic Tissue Clearing Method for Rat Brain Tissue at JoVE.com

这项工作由国家卫生研究院赠款资助：NS100624、DA013137、HD043680、MH106392 – T32 培训补助金 5T32GM081740

所有动物协议都经过南卡罗来纳大学动物护理和使用委员会的审查和批准。
1. 库存解决方案准备
<ol><li>解决方案 1 （1 L）： 到 900 mL 的去离子 H2O 添加 100 mL 的 10 倍磷酸盐缓冲盐水 （PBS） 和 2 mL 的 Triton X-100。</li>
	<li>解决方案 2 （1 L）： 至 900 mL 的去离子 H2O， 添加 100 mL 的 10 倍 PBS， 2mL 的 Tween-20 和 1 mL 的 10 毫克/mL 肝素库存解决方案。</li>
	<li>解决方案 3 ...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmental+distribution+of+ventral+striatal+neurons+projecting+to+the+mesencephalon+in+the+rat.">Compartmental distribution of ventral striatal neurons projecting to the mesencephalon in the rat.</a> Journal of Neuroscienc. 12 (6), 2079-2103 (1992).</li><li>Peyron, C., Petit, J. M., Rampon, C., Jouvet, M., Luppi, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forebrain+afferents+to+the+rat+dorsal+raphe+nucleus+demonstrated+by+retrograde+and+anterograde+tracing+methods.">Forebrain afferents to the rat dorsal raphe nucleus demonstrated by retrograde and anterograde tracing methods.</a> Neuroscience. 82 (2), 443-468 (1998).</li><li>Barrot, M., 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=Braking+dopamine+systems:+a+new+GABA+master+structure+for+mesolimbic+and+nigrostriatal+functions.">Braking dopamine systems: a new GABA master structure for mesolimbic and nigrostriatal functions.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscienc. 32 (41), 14094-14101 (2012).</li><li>Roostalu, U., 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=Quantitative+whole-brain+3D+imaging+of+tyrosine+hydroxylase-labeled+neuron+architecture+in+the+mouse+MPTP+model+of+Parkinson's+disease.">Quantitative whole-brain 3D imaging of tyrosine hydroxylase-labeled neuron architecture in the mouse MPTP model of Parkinson's disease.</a> Disease Models &amp; Mechanisms. 12 (11), (2019).</li><li>Ransohoff, R., Cardona, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myeloid+cells+of+the+central+nervous+system+parenchyma.">The myeloid cells of the central nervous system parenchyma.</a> Nature. 468, 253-262 (2010).</li><li>Rocha, M. 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组织清除为传统 IHC 协议的局限性提供了解决方案。透明的样品可最大限度地减少光的散射和吸收，从而提供细胞水平的光学访问到完整的组织18，19。组织清除技术通过脱毛、去装饰和贴花与溶剂的管理使组织透明化。一旦组织样本的折射指数与所选成像介质的折射索引匹配，样本将呈现完全透明，并可以正确成像3。目前的疏水方案?...

一些组织清除技术已被验证用于大脑：水凝胶，亲水性和疏水性。这些技术旨在通过溶剂的管理，通过脱毛、去装饰和贴花来使组织透明化。一旦组织样本的折射指数与所选成像介质的折射指数匹配，就可以获得样本的清晰图像。水凝胶技术，如 CLARITY，通过将生物分子与丙烯酸酯水凝胶连接起来，确保组织中的生物分子的安全，从而防止结构损伤和蛋白质损失1.然而，水凝胶技术使用的苛刻化学物质比有可能损害更脆弱的组织，更密集的组织样本可能不符合水凝胶协议。某些水凝胶技术可能需要昂贵的设备或导致组织扩张。亲水技术，如立方体，通过在组织2内形成氢键来保存3D结构。组织扩张也可能发生在某些亲水协议中。亲水技术的清除能力往往与疏水技术的能力不相称，这对更稠密、更厚的组织非常重要。
疏水技术通常速度快，不需要特殊设备，并产生易于处理和存储的样品。iDISCO 是一种疏水技术，可消除其他疏水协议中可能发生的收缩。最初，DISCO组织清除技术是由Renier等人描述的 ，用于小鼠的胚胎和密集的成人器官。该技术在初始脱水步骤中去除组织中的水，从而减少光散射。组织在预处理过程中渗透，允许深层抗体渗透。远红光谱中的亚历克萨氟染料用于免疫标签，以避免组织在低波长5的自身发光。经过疏水清除协议的组织能够轻松处理，并且由于 Alexa 氟染料的寿命长，可以成像几次。如果储存得当，组织可以在初始处理后提供数月到一年的图像。
在本协议中，酪氨酸羟基酶（TH）抗体、Iba1抗体和霍乱毒素亚细胞B（CTB）用于男性和女性控制以及HIV-1转基因（Tg）大鼠大脑。HIV-1 Tg大鼠拥有构成HIV-1病毒基因组的9个基因中的7个，这导致了长期HIV-1蛋白暴露的非传染性模型6，7，8。多巴胺变种以前在HIV-1 Tg大鼠身上已经证明，HIV-1本身是一种炎症性疾病，所以抗体选择与实验设计9，10，11，12有关。CTB是一种通过结核结合附着在神经元的示踪器，可用于追踪大脑中令人不关心的投影。CTB以前曾被用来研究从细胞核到亚斯坦蒂娅黑鬼区域的投影，大脑的两个区域严重涉及多巴胺性通路13、14、15。
在此协议中，TH 将作为多巴胺生产的标志，Iba1 将标记激活的微胶质。TH 抗体使用选择性地标记单个带在大约 62 kDa，对应酪氨酸羟基酶。Iba1 抗体对应于 Iba1 卡盒终端序列。这两种抗体都是在兔子身上培养的，是多克隆的。CTB 将用于逆行追踪神经元从细胞核区到亚斯坦蒂娅黑鬼区域。当前协议还以两种不同的方式为原 iDISCO 协议的调整提供了指导：1） 通过更改阻塞步骤中的血清试剂来全面减少背景染色，2） 扩大孵化时间以适合较大的组织样本。总体而言，本协议提供了持续的证据，证明疏水清除技术在大鼠的脑组织中是可行的，与HIV-1病毒蛋白没有明显的有害相互作用，并且与TH抗体、Iba1抗体和CTB相容。

<table>
	<tbody>
		<tr>
			<td>Cholera Toxin Subunit B (Recombinant), Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>C34775</td>
			<td></td>
		</tr>
		<tr>
			<td>DBE</td>
			<td>Sigma-Aldrich</td>
			<td>108014-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>DCM</td>
			<td>Sigma-Aldrich</td>
			<td>270997-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>472301-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher Chemical</td>
			<td>G46-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor Plus 647</td>
			<td>Invitrogen</td>
			<td>A32733</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma Life Science</td>
			<td>G9023-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Acros Organics</td>
			<td>41121-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Iba1 primary antibody</td>
			<td>FUJIFILM Wako</td>
			<td>019-19741</td>
			<td></td>
		</tr>
		<tr>
			<td>Kwik-sil epoxy</td>
			<td>VWR</td>
			<td>70730-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860-1l-R</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Bioreagents</td>
			<td>BP2944-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion machine</td>
			<td>VWR</td>
			<td>70730-062</td>
			<td>mini pump variable flow</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Sigma-Aldrich</td>
			<td>158127-3KG</td>
			<td></td>
		</tr>
		<tr>
			<td>TH primary antibody</td>
			<td>Millipore Sigma</td>
			<td>AB152</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Fisher Bioreagents</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher Bioreagents</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

a hydrophobic tissue clearing method for rat brain tissue

疏水组织清除方法易于调节、快速和低成本的程序，允许研究未受变组织样本中感兴趣的分子。传统的免疫标签程序要求将样品切成薄部分，这限制了标记和检查完整结构的能力。但是，如果脑组织在处理过程中能够保持完整，则结构和回路可以保持完好无损进行分析。先前建立的清除方法需要大量时间才能完全清除组织，而苛刻的化学物质通常会损害敏感的抗体。iDISCO 方法快速、完全地清除组织，与许多抗体兼容，无需特殊的实验室设备。这项技术最初被验证用于小鼠组织，但目前的协议使这种方法适应了控制半球和转基因大鼠大脑的图像。除此之外，本协议还对预先存在的协议进行了若干调整，以提供更清晰的图像，减少背景污渍。使用目前的疏水组织清除方法，在HIV-1转基因大鼠和F344/N控制大鼠中验证了Iba-1和酪氨酸羟基酶的抗体。大脑是一个交织在一起的网络，结构相互作用的频率高于彼此的相互作用。分析整个系统的大脑，而不是单个片段的组合，是整个大脑清理方法的最大好处。

利用这种经过修改的疏水组织清除方案，实现了F344/N和HIV-1大鼠脑组织的大面积完全清除。 图1 显示了在亚斯坦蒂娅尼格拉区域TH的典型共聚焦图像。 图 1A 表示密集、正色。通过"Z"平面聚焦以确认正染色和适当的细胞形态，可以分析出像这样的密集区域。 图1B 表示稀疏的正染色，可以通过TH神经元独特的形态来识别。 <strong class="...

Watch this Scientific Journal Video about A Hydrophobic Tissue Clearing Method for Rat Brain Tissue at JoVE.com

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

在这里，我们提出了一个疏水组织清除方法，允许查看目标分子作为完整的大脑结构的一部分。这项技术现已得到F344/N控制和艾滋病毒-1转基因两性大鼠的验证。

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 ...

Research

JoVE Journal

Neuroscience

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

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 ...

科研

神经科学

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

脑植入的微型器件及周围组织的完整的组织学特性研究

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

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 ...

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 ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

DAN矩阵升华为神经节苷脂对大鼠脑组织的检测和可视化MALDI成像质谱

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

轻度宫内灌注圈狭窄的大鼠模型

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

一种新的用于全中枢神经系统光透明快速生成的无源清除方法

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

新鲜人脑组织中毛细血管的分离

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...