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

This study was supported by the China Academy of Chinese Medical Sciences Innovation Fund (Project Code no. CI2021A03404) and the National Traditional Chinese Medicine Interdisciplinary innovation Fund (Project Code no. ZYYCXTD-D-202202).

The present study was approved by the Ethics Committee of the Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences (reference number D2018-04-13-1). All procedures were conducted following the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C., 1996). Three adult male rats (Sprague-Dawley, weight 230 &#177; 15 g) were used in this study. All animals were housed in a 12 h light/dark cycle with controlled temperature and hu...

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The present study provides a detailed demonstration of the cutaneous nerve fibers in the hairy and glabrous skin by using immunofluorescence on thicker tissue sections with clearing treatment and a 3D view to understand the skin innervation better. The antibody incubation time of up to 1-2 days and an overnight cleaning process are important. These two key steps directly affect the immunofluorescence staining effect of thick sections. A further problem was raised from the choice of antibodies, not all of which are suitab...

The skin, the largest organ in the body, serving as a key interface to the environment, is densely innervated by many nerve fibers1,2,3. Although skin innervation has been widely studied previously with various histological methods, such as staining on whole-mount skin and tissue sections4,5,6, the detailed effective demonstration of cutaneous nerve fibers is still a challenge7,8. Given this, the present protocol developed a unique technique to exhibit cutaneous nerve fibers more clearly in the thick tissue section.
Because of the limit by the thickness of sections, the observation of innervated skin nerve fibers is not precise enough to accurately depict the relationship between calcitonin gene-related peptide (CGRP) nerve fibers and local tissues and organs from the acquired image information. The emergence of 3D tissue clearing technology provides a feasible method to solve this problem9,10. The fast development of tissue-clearing approaches has offered many tools for studying tissue structures, entire organs, neuronal projections, and whole animals in recent times11. The transparent skin tissue could be imaged in a much thicker section by confocal microscopy to obtain the data to visualize cutaneous nerve fibers.
In the current study, the plantar and dorsal skin of a rat hindfoot was selected as the two target sites of hairy and glabrous skin3,4,7. To trace the cutaneous nerve fibers at a longer distance, the skin tissue was sliced at the thickness of 300 &#181;m for immunohistochemical and histochemical staining, followed by tissue clearing treatment. CGRP was used to label the sensory nerve fibers12,13. In addition, to highlight the cutaneous nerve fibers on the tissue background, phalloidin and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) were further used to label the blood vessels and lymphatic vessels, respectively14,15.
These approaches provided a straightforward method that can be applied to demonstrate a high-resolution view of the cutaneous nerve fibers and also to visualize the spatial correlation among the nerve fibers, blood vessels, and lymphatic vessels in the skin, which may provide much more information to understand the homeostasis of the normal skin and the cutaneous alteration under the pathological conditions.

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	<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

Skin innervation is an important part of the peripheral nervous system. Although the study of the cutaneous nerve fibers has progressed rapidly, most of the understanding of their distributional and chemical characteristics comes from conventional histochemical and immunohistochemical staining on thin tissue sections. With the development of the tissue clearing technique, it has become possible to view the cutaneous nerve fibers on thicker tissue sections. The present protocol describes multiple fluorescent staining on tissue sections at a thickness of 300 &#181;m from the plantar and dorsal skin of rat hindfoot, the two typical hairy and glabrous skin sites. Here, the calcitonin gene-related peptide labels the sensory nerve fibers, while phalloidin and lymphatic vessel endothelial hyaluronan receptor 1 label the blood and lymphatic vessels, respectively. Under a confocal microscope, the labeled sensory nerve fibers were followed completely at a longer distance, running in bundles in the deep cutaneous layer and freestyle in the superficial layer. These nerve fibers ran in parallel to or surrounded the blood vessels, and lymphatic vessels formed a three-dimensional (3D) network in the hairy and glabrous skin. The current protocol provides a more effective approach to studying skin innervation than the existing conventional methods from the methodology perspective.

After triple fluorescent staining, the nerve fibers, blood vessels, and lymphatic vessels were clearly labeled with CGRP, phalloidin, and LYVE1, respectively, in the hairy and glabrous skin (Figure 3,4). With the clearing treatment, the CGRP-positive nerve fibers, phalloidin-positive blood vessels, and LYVE1-positive lymphatic vessels can be imaged at a greater depth to acquire the complete structural information of the skin (Figure 3). When the...

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

The thickness of tissue sections limited the morphological study of the skin innervation. The present protocol describes a unique tissue clearing technique to visualize cutaneous nerve fibers in thick 300 &#181;m tissue sections under confocal microscopy.

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

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