Name: demonstrating hairy and glabrous skin innervation in a 3d pattern using multiple fluorescent staining and tissue clearing approaches
Uploaded: 2022-05-20T14:00:00
Description: Watch this Scientific Journal Video about Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches at JoVE.com

本研究由中国中医科学院创新基金（项目代码：20199）资助。CI2021A03404）和国家中医药交叉创新基金（项目代码：2021A03404）。ZYYCXTD-D-202202）.。

本研究经中国中医科学院针灸研究所伦理学委员会批准（参考编号D2018-04-13-1）。所有程序都是根据《国家卫生研究院实验动物护理和使用指南》（国家科学院出版社，华盛顿特区，1996年）进行的。本研究使用了三只成年雄性大鼠（斯普拉格 - 道利，体重230±15g）。所有动物都被饲养在12小时的光/暗循环中，温度和湿度受控，并允许自由获取食物和水。
1. 灌注和样品制备<...

<ol>
	<li>Vidal Yucha, S. E., Tamamoto, K. A., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+the+neuro-immuno-cutaneous+system+on+human+skin+equivalent+design.">The importance of the neuro-immuno-cutaneous system on human skin equivalent design.</a> Cell Proliferation. 52 (6), 12677 (2019).</li><li>Wu, H., Williams, J., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+diversity+of+cutaneous+sensory+afferents+revealed+by+genetically+directed+sparse+labeling.">Morphologic diversity of cutaneous sensory afferents revealed by genetically directed sparse labeling.</a> Elife. 1, 00181 (2012).</li><li>Pomaville, M. B., Wright, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+and+genetic+labeling+of+hairy+and+glabrous+skin+innervation.">Immunohistochemical and genetic labeling of hairy and glabrous skin innervation.</a> Currents Protocols. 1 (5), 121 (2021).</li><li>Navarro, X., Verd&#250;, E., Wendelscafer-Crabb, G., Kennedy, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innervation+of+cutaneous+structures+in+the+mouse+hind+paw:+a+confocal+microscopy+immunohistochemical+study.">Innervation of cutaneous structures in the mouse hind paw: a confocal microscopy immunohistochemical study.</a> Journal of Neuroscience Research. 41 (1), 111-120 (1995).</li><li>Chang, H., Wang, Y., Wu, H., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flat+mount+imaging+of+mouse+skin+and+its+application+to+the+analysis+of+hair+follicle+patterning+and+sensory+axon+morphology.">Flat mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology.</a> Journal of Visualized Experiments. (88), e51749 (2014).</li><li>Yamazaki, T., Li, W., Mukouyama, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+confocal+microscopy+for+adult+ear+skin:+a+model+system+to+study+neuro-vascular+branching+morphogenesis+and+immune+cell+distribution.">Whole-mount confocal microscopy for adult ear skin: a model system to study neuro-vascular branching morphogenesis and immune cell distribution.</a> Journal of Visualized Experiments. (133), e57406 (2018).</li><li>Hendrix, S., Picker, B., Liezmann, C., Peters, 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=Skin+and+hair+follicle+innervation+in+experimental+models:+a+guide+for+the+exact+and+reproducible+evaluation+of+neuronal+plasticity.">Skin and hair follicle innervation in experimental models: a guide for the exact and reproducible evaluation of neuronal plasticity.</a> Experimental Dermatology. 17 (3), 214-227 (2008).</li><li>Salz, L., Driskell, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+whole+mount:+a+novel+processing+and+imaging+protocol+for+thick,+three-dimensional+tissue+cross-sections+of+skin.">Horizontal whole mount: a novel processing and imaging protocol for thick, three-dimensional tissue cross-sections of skin.</a> Journal of Visualized Experiments. (126), e56106 (2017).</li><li>Yokomizo, T., 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=Whole-mount+three-dimensional+imaging+of+internally+localized+immunostained+cells+within+mouse+embryos.">Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos.</a> Nature Protocols. 7 (3), 421-431 (2012).</li><li>Jing, 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=Tissue+clearing+of+both+hard+and+soft+tissue+organs+with+the+PEGASOS+method.">Tissue clearing of both hard and soft tissue organs with the PEGASOS method.</a> Cell Research. 28 (8), 803-818 (2018).</li><li>Pende, 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=High-resolution+ultramicroscopy+of+the+developing+and+adult+nervous+system+in+optically+cleared+Drosophila+melanogaster.">High-resolution ultramicroscopy of the developing and adult nervous system in optically cleared Drosophila melanogaster.</a> Nature Communications. 9 (1), 4731 (2018).</li><li>Russell, F. A., King, R., Smillie, S. J., Kodji, X., Brain, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcitonin+gene-related+peptide:+physiology+and+pathophysiology.">Calcitonin gene-related peptide: physiology and pathophysiology.</a> Physiological Reviews. 94 (4), 1099-1142 (2014).</li><li>Wang, J., 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=Visualizing+the+calcitonin+gene-related+peptide+immunoreactive+innervation+of+the+rat+cranial+dura+mater+with+immunofluorescence+and+neural+tracing.">Visualizing the calcitonin gene-related peptide immunoreactive innervation of the rat cranial dura mater with immunofluorescence and neural tracing.</a> Journal of Visualized Experiments. (167), e61742 (2021).</li><li>Wulf, E., Deboben, A., Bautz, F. A., Faulstich, H., Wieland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+phallotoxin,+a+tool+for+the+visualization+of+cellular+actin.">Fluorescent phallotoxin, a tool for the visualization of cellular actin.</a> Proceedings of the National Academy of Sciences of the United States of America. 76 (9), 4498-4502 (1979).</li><li>Banerji, 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=LYVE-1,+a+new+homologue+of+the+CD44+glycoprotein,+is+a+lymph-specific+receptor+for+hyaluronan.">LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.</a> Journal of Cell Biology. 144 (4), 789-801 (1999).</li><li>Susaki, E. 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=Advanced+CUBIC+protocols+for+whole-brain+and+whole-body+clearing+and+imaging.">Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.</a> Nature Protocols. 10 (11), 1709-1727 (2015).</li><li>Liang, H., 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=CUBIC+protocol+visualizes+protein+expression+at+single+cell+resolution+in+whole+mount+skin+preparations.">CUBIC protocol visualizes protein expression at single cell resolution in whole mount skin preparations.</a> Journal of Visualized Experiments. (114), e54401 (2016).</li><li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues.">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nature Protocols. 9 (7), 1682-1697 (2014).</li><li>Cai, R., 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=Panoptic+imaging+of+transparent+mice+reveals+whole-body+neuronal+projections+and+skull-meninges+connections.">Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections.</a> Nature Neuroscience. 22 (2), 317-327 (2019).</li><li>Ashrafi, M., Baguneid, M., Bayat, 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+role+of+neuromediators+and+innervation+in+cutaneous+wound+healing.">The role of neuromediators and innervation in cutaneous wound healing.</a> Acta Dermato-Venereologica. 96 (5), 587-594 (2016).</li><li>Wang, J., 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=A+new+approach+for+examining+the+neurovascular+structure+with+phalloidin+and+calcitonin+gene-related+peptide+in+the+rat+cranial+dura+mater.">A new approach for examining the neurovascular structure with phalloidin and calcitonin gene-related peptide in the rat cranial dura mater.</a> Journal of Molecular Histology. 51 (5), 541-548 (2020).</li><li>Chazotte, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+cytoskeletal+F-actin+with+rhodamine+phalloidin+or+fluorescein+phalloidin+for+imaging.">Labeling cytoskeletal F-actin with rhodamine phalloidin or fluorescein phalloidin for imaging.</a> Cold Spring Harbor Protocols. (5), (2010).</li><li>Breslin, J. W., 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=Lymphatic+vessel+network+structure+and+physiology.">Lymphatic vessel network structure and physiology.</a> Comprehensive Physiology. 9 (1), 207-299 (2018).</li><li>Schwager, S., Detmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+lymphatic+function.">Inflammation and lymphatic function.</a> Frontiers in Immunology. 10, 308 (2019).</li><li>Hagan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+fission+yeast+filamentous+actin+with+fluorescent+phalloidin+conjugates.">Staining fission yeast filamentous actin with fluorescent phalloidin conjugates.</a> Cold Spring Harbor Protocol. (6), (2016).</li></ol>

本研究通过在较厚的组织切片上使用免疫荧光进行清除处理和3D视图以更好地了解皮肤神经支配，详细演示了毛茸茸和光滑皮肤中的皮肤神经纤维。长达1-2天的抗体孵育时间和过夜清洁过程非常重要。这两个关键步骤直接影响厚切片的免疫荧光染色效果。抗体的选择提出了另一个问题，并非所有抗体都适用于厚切片。我们推测，分子量较小的抗体可能是厚切片免疫荧光染色的理想选择。因此，抗?...

皮肤是人体最大的器官，作为环境的关键界面，由许多神经纤维1，2，3密集支配。尽管皮肤神经支配以前已经通过各种组织学方法进行了广泛的研究，例如在全贴片皮肤和组织切片上染色4，5，6，但皮肤神经纤维的详细有效演示仍然是一个挑战7，8。鉴于此，本方案开发了一种独特的技术，可以在厚组织切片中更清楚地显示皮肤神经纤维。
由于切片厚度的限制，对受支配的皮肤神经纤维的观察不够精确，无法从获得的图像信息中准确描述降钙素基因相关肽（CGRP）神经纤维与局部组织和器官之间的关系。3D组织清除技术的出现为解决这一问题提供了一种可行的方法9，10。近年来，组织清除方法的快速发展为研究组织结构，整个器官，神经元投影和整个动物提供了许多工具11.透明的皮肤组织可以通过共聚焦显微镜在更厚的部分成像，以获得可视化皮肤神经纤维的数据。
在目前的研究中，选择大鼠后足的足底和背侧皮肤作为毛茸茸和光滑皮肤的两个靶位点3，4，7。为了在更长的距离上追踪皮肤神经纤维，将皮肤组织切片在300μm的厚度下进行免疫组织化学和组织化学染色，然后进行组织清除处理。CGRP用于标记感觉神经纤维12，13。此外，为了突出组织背景上的皮肤神经纤维，还进一步使用鬼笔分析蛋白和淋巴管内皮透明质酸受体1（LYVE1）分别标记血管和淋巴管14，15。
这些方法提供了一种简单的方法，可用于展示皮肤神经纤维的高分辨率视图，并可视化皮肤中神经纤维，血管和淋巴管之间的空间相关性，这可能为了解正常皮肤的稳态和病理条件下的皮肤改变提供更多的信息。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x phosphate-buffered saline</td>
			<td>Solarbio Life Sciences</td>
			<td>P1020</td>
			<td>pH 7.2-7.4, 0.01 Mol</td>
		</tr>
		<tr>
			<td>2,2,2-Tribromoethanol</td>
			<td>Sigma Life Science</td>
			<td>T48402-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal fluorescence microscopy</td>
			<td>Olympus Corporation</td>
			<td>Fluoview FV1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-mouse IgG H&#38;L Alexa-Flour488</td>
			<td>Abcam plc.</td>
			<td>ab150105</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-sheep IgG H&#38;L Alexa-Flour405</td>
			<td>Abcam plc.</td>
			<td>ab175676</td>
			<td></td>
		</tr>
		<tr>
			<td>EP tube</td>
			<td>Wuxi NEST Biotechnology Co.</td>
			<td>615001</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Freezing stage sliding microtome system</td>
			<td>Leica Biosystems</td>
			<td>CM1860</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris Software</td>
			<td>Oxford Instruments</td>
			<td>v.9.0.1</td>
			<td></td>
		</tr>
		<tr>
			<td>IRIS standard scissor</td>
			<td>WPI (World Precision Instruments Inc.)</td>
			<td>503242</td>
			<td></td>
		</tr>
		<tr>
			<td>iSpacer</td>
			<td>SunJin Lab co.</td>
			<td>IS005</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro forceps-Str</td>
			<td>RWD</td>
			<td>F11020-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-CGRP antibody</td>
			<td>Santa cruz biotechnology, Inc.</td>
			<td>sc-57053</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral buffered Formalin</td>
			<td>Solarbio Life Sciences</td>
			<td>G2161</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>017-000-12</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Longer Precision Pump Co., Ltd</td>
			<td>BT300-2J</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin Alexa-Fluor 594</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12381</td>
			<td></td>
		</tr>
		<tr>
			<td>RapiClear 1.52 solution</td>
			<td>SunJin Lab co.</td>
			<td>RC152001</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Regular agarose</td>
			<td>Gene Company Limited</td>
			<td>G-10</td>
			<td></td>
		</tr>
		<tr>
			<td>SEMKEN 1 x 2 Teeth Tissue Forceps-Str</td>
			<td>RWD</td>
			<td>F13038-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep polyclonal anti-LYVE1 antibody</td>
			<td>R&#38;D Systems, Inc.</td>
			<td>AF7939</td>
			<td></td>
		</tr>
		<tr>
			<td>Six-well plate</td>
			<td>Corning Incorporated</td>
			<td>3335</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma Life Science</td>
			<td>S2002</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Life Science</td>
			<td>V900116</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Super Glue</td>
			<td>Henkel AG &#38; Co.</td>
			<td>Pattex 502</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Handles</td>
			<td>RWD</td>
			<td>S32003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Solarbio Life Sciences</td>
			<td>9002-93-1</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Urethane</td>
			<td>Sigma Life Science</td>
			<td>U2500</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>VANNAS spring scissors</td>
			<td>RWD</td>
			<td>S1014-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratory microtome</td>
			<td>Leica Biosystems</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstrating hairy and glabrous skin innervation in a 3d pattern using multiple fluorescent staining and tissue clearing approaches

皮肤神经支配是周围神经系统的重要组成部分。虽然对皮肤神经纤维的研究进展迅速，但对其分布和化学特性的大部分理解来自对薄组织切片的常规组织化学和免疫组织化学染色。随着组织清除技术的发展，已经可以在较厚的组织切片上观察皮肤神经纤维。本方案描述了从大鼠后足的足底和背侧皮肤（两个典型的毛茸茸和光滑的皮肤部位）以300μm的厚度对组织切片进行多次荧光染色。这里，降钙素基因相关肽标记感觉神经纤维，而鬼臼肽和淋巴管内皮透明质酸受体1分别标记血液和淋巴管。在共聚焦显微镜下，标记的感觉神经纤维在更长的距离上完全跟随，在深层皮肤层成束运行，在浅层自由泳。这些神经纤维平行于或包围血管，淋巴管在毛茸茸的皮肤中形成三维（3D）网络。从方法论的角度来看，目前的方案提供了一种比现有传统方法更有效的研究皮肤神经支配的方法。

三重荧光染色后，神经纤维，血管和淋巴管分别在毛茸茸和光滑的皮肤中用CGRP，鬼臼素和LYVE1清楚地标记（图3，4）。通过清除治疗，可以在更深的深度对CGRP阳性神经纤维，鬼臼肽阳性血管和LYVE1阳性淋巴管进行成像，以获得皮肤的完整结构信息（图3）。当这些组织结构以3D模式进一步重建时，它们的分布变得更容易追踪。结果表明，CGRP?...

Watch this Scientific Journal Video about Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches at JoVE.com

Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches

组织切片的厚度限制了皮肤神经支配的形态学研究。本方案描述了一种独特的组织清除技术，用于在共聚焦显微镜下可视化300μm厚组织切片中的皮肤神经纤维。

使用多种荧光染色和组织清除方法以3D模式展示毛茸茸和光滑的皮肤神经支配

Skin innervation is an important part of the peripheral nervous system. Although the study of the cutaneous nerve fibers has progressed rapidly, most of ...

该方案提供了一种使用共聚焦显微镜观察同步部分的可行方法，可以确保神经纤维观察的精度。简单易用的组织清洁方案是这种技术的主要优点。组织清洁过程非常高效，大约需要30分钟到一个小时。如文本手稿中所述，对安乐死的大鼠进行灌注。使用手术刀从后爪上取下背部和鞋底的皮肤。对于需要冷冻切片的样品，将组织从4%的醛中固定在0.1摩尔PB中两小时。洗涤后，将皮肤组织嵌入37摄氏度的2%琼脂糖中，并将其放入冰盒中冷却。用冰水将安装的组织固定在振动微调上，并在横向方向上以500微米的厚度将其切片。切片后，从切片中取出琼脂糖并将其储存在具有1x PBS的六孔板中。将组织切片在2%曲肽X-100的溶液中在1x PBS中以4摄氏度孵育过夜。将组织切片放入封闭缓冲液中，并在振荡器上以每分钟72次旋转，在四摄氏度下旋转过夜。将组织切片转移到含有小鼠单克隆抗CGRP和绵羊多克隆抗LYVE-1抗体的一抗的溶液中，在微量离心管中的稀释缓冲液中。然后在摇床上以四摄氏度旋转两天。在室温下用洗涤缓冲液洗涤组织切片两次，然后在洗涤缓冲液中以四摄氏度将其保持在振荡器上过夜。第二天，将组织切片转移到微量离心管中含有二抗的混合溶液中，并在振荡器上以每分钟72转的速度在四摄氏度下旋转五小时。在室温下用洗涤缓冲液在六孔板中洗涤组织切片两次，持续一小时。将组织切片保持在振荡器上的洗涤缓冲液中，在四摄氏度下过夜。将组织切片转移到组织清除试剂中，并在室温下在振荡器上以每分钟60转的速度轻轻旋转一小时。将透明组织安装在载玻片上，然后用垫片圈住组织，并用新鲜的组织清除试剂和盖玻片粘住间隙。3D模式表明CGRP阳性神经纤维通过皮下组织和真皮到达表皮。这些神经纤维在皮下组织中成束运行，在真皮内分支并终止于表皮。相反，阳性血管中的鬼臼蛋白和LYVE-1阳性淋巴管分布在皮下组织和真皮中。通常，CGRP阳性神经纤维平行或包围血管和淋巴管，在毛茸茸和光滑的皮肤中形成3D网络。使用常规方法，虽然神经纤维血管和淋巴管在薄片中用CGRP鬼笔环蛋白和LYVE-1清楚地标记，但这些结构的观察受到切片厚度的限制，无法完全观察到。在厚度为30 μm的切片中，CGRP阳性神经纤维表面积的毛茸茸的皮肤和光滑的皮肤之间没有差异。在300微米厚的组织切片中，与毛茸茸的皮肤相比，松弛皮肤CGRP正神经纤维的表面积显著增加。在抗体孵育后使用具有高渗透压的清洁缓冲液非常重要。几次，免疫荧光染色所需的清洁时间是必要的。按照这个程序，我们认为有可能染色和观察其他组织和器官的六个部分也存在。这种技术可以帮助神经科学研究人员了解神经纤维的表面积和状态。它还有助于我们观察组织和器官的创新。

Research

JoVE Journal

Neuroscience

Cross-Modal Multivariate Pattern Analysis

跨模态的多元格局分析

Multivariate pattern analysis (MVPA) is an increasingly popular method of analyzing functional magnetic resonance imaging (fMRI) data1-4. Typically, the ...

科研

神经科学

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

鼠标感觉神经支配的皮肤和四肢骨骼纤维组织的制备及免疫染色

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

脑室周围组织的侧脑室及组织学特性对人类和鼠标3D建模

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

微流控采用共培养开发设备牙胚支配分析

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

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, ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

基于改进多平台法的大鼠中心疲劳模型

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

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 ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

在体内成像完全活跃的脑组织在清醒斑马鱼幼虫和青少年由骷髅和皮肤去除

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...