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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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<…
Representative Results
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?…
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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）.。
Materials
| 1x phosphate-buffered saline | Solarbio Life Sciences | P1020 | pH 7.2-7.4, 0.01 Mol |
| 2,2,2-Tribromoethanol | Sigma Life Science | T48402-5G | |
| Confocal fluorescence microscopy | Olympus Corporation | Fluoview FV1200 | |
| Donkey anti-mouse IgG H&L Alexa-Flour488 | Abcam plc. | ab150105 | |
| Donkey anti-sheep IgG H&L Alexa-Flour405 | Abcam plc. | ab175676 | |
| EP tube | Wuxi NEST Biotechnology Co. | 615001 | 1.5 mL |
| Freezing stage sliding microtome system | Leica Biosystems | CM1860 | |
| Imaris Software | Oxford Instruments | v.9.0.1 | |
| IRIS standard scissor | WPI (World Precision Instruments Inc.) | 503242 | |
| iSpacer | SunJin Lab co. | IS005 | |
| Micro forceps-Str | RWD | F11020-11 | |
| Mouse monoclonal anti-CGRP antibody | Santa cruz biotechnology, Inc. | sc-57053 | |
| Neutral buffered Formalin | Solarbio Life Sciences | G2161 | 10% |
| Normal donkey serum | Jackson ImmunoResearch Laboratories | 017-000-12 | 10 mL |
| Peristaltic pump | Longer Precision Pump Co., Ltd | BT300-2J | |
| Phalloidin Alexa-Fluor 594 | Thermo Fisher Scientific | A12381 | |
| RapiClear 1.52 solution | SunJin Lab co. | RC152001 | 10 mL |
| Regular agarose | Gene Company Limited | G-10 | |
| SEMKEN 1 x 2 Teeth Tissue Forceps-Str | RWD | F13038-12 | |
| Sheep polyclonal anti-LYVE1 antibody | R&D Systems, Inc. | AF7939 | |
| Six-well plate | Corning Incorporated | 3335 | |
| Sodium azide | Sigma Life Science | S2002 | 25 g |
| Sucrose | Sigma Life Science | V900116 | 500 g |
| Super Glue | Henkel AG & Co. | Pattex 502 | |
| Surgical Handles | RWD | S32003-12 | |
| Triton X-100 | Solarbio Life Sciences | 9002-93-1 | 100 mL |
| Urethane | Sigma Life Science | U2500 | 500 g |
| VANNAS spring scissors | RWD | S1014-12 | |
| Vibratory microtome | Leica Biosystems | VT1200S |
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Citar este artigo
Wang, X., Cao, W., Shi, J., Zhang, X., Qu, Z., Xu, D., Wan, H., Su, Y., He, W., Jing, X., Bai, W. Demonstrating Hairy and Glabrous Skin Innervation in a 3D Pattern Using Multiple Fluorescent Staining and Tissue Clearing Approaches. J. Vis. Exp. (183), e63807, doi:10.3791/63807 (2022).