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 humidity and allowed free access to food and water.
1. Perfusion and sample preparation
<ol>
	<li>Inject 250 mg/kg of tribromoethanol solution (see Table of Materials) intraperitoneally into the rat to induce euthanasia.</li>
	<li>Once breathing stops, use stainless steel surgical scissors to open the thoracic cavity of the rat. Use 20 G needles to perfuse via the left cardiac ventricle13 at a rate of 3 mL/min with 100 mL of 0.9% normal saline followed by 250-300 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) (Figure 1A,B).</li>
	<li>After perfusion, use a scalpel to remove the skin of the dorsum and sole from the hind paw.
	<ol>
		<li>For the samples requiring frozen section, post-fix the tissues in 4% paraformaldehyde in 0.1 M PB for 2 h; then cryoprotect the tissues in 25% sucrose in 0.1 M PB for more than 24 h at 4 &#176;C</li>
		<li>For the samples with the thick section that requires tissue clearing, post-fix the tissues in 4% paraformaldehyde in 1x phosphate-buffered saline (1x PBS) for 2 h; then cryoprotect the tissues in 1x PBS at 4 &#176;C (Figure 1C&#8211;E).</li>
	</ol>
	</li>
</ol>
2. Triple fluorescent staining with CGRP, phalloidin, and LYVE1 followed by tissue clearing treatment
NOTE: Triple fluorescent staining with CGRP, phalloidin and LYVE1 was applied to reveal the nerve fibers, blood vessels, and lymphatic vessels in hairy and glabrous skin on tissue sections with a thickness of 300 &#181;m following tissue clearing treatment.
<ol>
	<li>Prepare 2% agarose in 1x&#160;PBS by heating in a micro-oven for 2 min until the agarose dissolves (see&#160;Table of Materials).</li>
	<li>After washing, embed the skin tissues into 2% agarose at 37 &#176;C, and place them inside the icebox for cooling (Figure 1F,G).</li>
	<li>Fix the mounted tissue on the vibratory microtome with the ice water and slice them at a thickness of 500 &#181;m in the transversal direction. Use the following parameters to set the vibration slicer and finish slicing: speed, 0.50 mm/s, and amplitude, 1.5 mm (Figure 1H-K).</li>
	<li>After slicing, remove the agarose from the sections, and store them in a six-well plate with 1x&#160;PBS (pH 7.4) (Figure 1L).</li>
	<li>Incubate the tissue sections in a solution of 2% Triton X-100 in 1x&#160;PBS (TriX-PB) overnight at 4 &#176;C. See Figure 2 for the following procedure.</li>
	<li>Place the tissue sections into the blocking buffer and rotate at 72 rpm on the shaker overnight at 4 &#176;C. 
	NOTE: The blocking buffer composition is 10% normal donkey serum, 1% Triton-X 100, and 0.2% sodium azide in 1x&#160;PBS (see Table of Materials).</li>
	<li>Transfer the tissue sections into the solution containing the primary antibodies of mouse monoclonal anti-CGRP (1:500) and sheep polyclonal anti-LYVE1 antibody (1:500) in dilution buffer in the microcentrifuge tube (see Table of Materials), and rotate on the shaker for 2 days at 4 &#176;C. 
	NOTE: The dilution buffer composition is 1% normal donkey serum, 0.2% Triton-X 100, and 0.2% sodium azide in 1x&#160;PBS.</li>
	<li>Wash the tissue sections twice with washing buffer at room temperature, then keep them on the shaker overnight at 4 &#176;C in washing buffer. 
	NOTE: The washing buffer composition is 0.2% Triton-X 100 in 0.1 M PB (pH 7.4).</li>
	<li>The next day, transfer the tissue sections into the mixed solution containing the secondary antibodies of donkey anti-mouse IgG H&#38;L Alexa-Flour488 (1:500) and donkey anti-sheep IgG H&#38;L Alexa-Flour405 (1:500), as well as Alexa-Flour594 phalloidin (1:1000) (see Table of Materials) in a microcentrifuge tube and rotate at 72 rpm on the shaker for 5 h at 4 &#176;C.</li>
	<li>Wash the tissue sections in a six-well plate with washing buffer for 1 h, twice, at room temperature. Keep the tissue sections in washing buffer on the shaker overnight at 4 &#176;C.</li>
	<li>Transfer the tissue sections into the tissue clearing reagent (see Table of Materials, its volume was five times more than the sample volume) and rotate at 60 rpm on the shaker gently for 1 h at room temperature. 
	NOTE: After this treatment, the tissue sections become clear (Figure 2B).</li>
	<li>Mount the cleared tissues on the slide, circle the tissues with a spacer, and stick the gap with fresh tissue clearing reagent and coverslip.</li>
</ol>
3. Triple fluorescent staining with CGRP, phalloidin, and LYVE1 following the conventional approach
NOTE: As a comparison, the same staining was performed on tissue sections at a thickness of 30 &#181;m following conventional techniques.
<ol>
	<li>Slice the tissue sections at 30 &#181;m on a microtome in the transversal direction and mount them on the slide.</li>
	<li>Add blocking solution with 3% normal donkey serum and 0.3% Triton X-100 in 0.1 M PB and incubate the sections for 30 min at room temperature.</li>
	<li>Remove the blocking solution and incubate the sections with the solution containing the primary antibodies of mouse monoclonal anti-CGRP (1:500) and sheep polyclonal anti-LYVE1 antibody (1:500) in dilution buffer overnight at 4 &#176;C.</li>
	<li>The next day, wash the tissue sections with 0.1 M PB (pH 7.4) three times, add the mixed solution containing the secondary antibodies of donkey anti-mouse IgG H&#38;L Alexa-Flour488 (1:500) and donkey anti-sheep IgG H&#38;L Alexa-Flour405 (1:500), as well as Alexa-Flour594 phalloidin (1:1000) on the sections and incubate for 1 h at room temperature.</li>
	<li>Before microscopic observation, wash the sections in 0.1 M PB (pH 7.4) three times, then cover them with coverslips in 50% glycerin.</li>
</ol>
4. Imaging and analyses
<ol>
	<li>Observe the stained samples under a fluorescent microscope, and then take the images using a confocal microscope.</li>
	<li>Capture 30 images (Z-stacks), each in 10 &#181;m frames, from each 300 &#181;m thick section and integrate a single in-focus image using an image processor of the confocal microscopy system (see Table of Materials).
	<ol>
		<li>Perform the following steps in the image processing software of the confocal setup for three-dimensional (3D) analysis: Set Start Focal Plane | Set End focal plane | Set step size | Choose Depth Pattern | Image Capture | Z series. 
		NOTE: The excitation and emission wavelengths of blue fluorescence signals were 401 nm and 421 nm, respectively. Excitation and emission wavelengths of green fluorescence signals were 499 nm and 519 nm, respectively. Excitation and emission wavelengths of red fluorescence signals were 591 nm and 618 nm, respectively. 152 &#181;m is the diameter of the confocal pinhole (10x objective lens, NA: 0.4). The image capture resolution was 1024 &#215; 1024 pixels.</li>
	</ol>
	</li>
	<li>For the conventional samples (step 3), capture 30 images (Z-stacks) in 1 &#181;m frames from each 30 &#181;m thick section and further treat these images as mentioned above (steps 4.1-4.2).</li>
	<li>Demonstrate the images in the pattern of 3D reconstruction with the image processing system.</li>
	<li>Perform 3D reconstructions by importing Z-stack confocal images into Imaris 9.0 (cell imaging software, see Table of Materials) and create surface renderings based on stain intensities: Add new surfaces | Choose Source Channel | Determine the parameters in the smooth option of Surfaces Detail | Determine the parameters in the threshold option of Absolute Intensity | Classify Surfaces | Finish.</li>
	<li>Acquire data in the surface area of positive nerve fibers with the cell imaging software. Select the rendered image | Statistics | Detailed | Specific Values | Volume.</li>
</ol>

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The authors have nothing to disclose.

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 suitable for thick sections. We speculate that antibodies with smaller molecular weights might be ideal for immunofluorescence staining of thick sections. Thus, the difficulty of antibody selection is a major limitation of this approach. The present work had observed the differences in the distribution of blood vessels, lymphatic vessels, and nerves on hairy and hairless skin of rats. For humans, the skin structure is very different from that of rodents; we look forward to using tissue clearing technology to observe and research human skin in the next step.
Previous studies have made many efforts to reveal cutaneous nerve fibers4,5,6,7,8. In contrast to the conventional tissue section study, tissue clearing techniques including CUBIC, CLARITY, and vDISCO have been widely used for studying entire organs and whole bodies of animals in recent years16,17,18,19. Usually, it is difficult to trace the cutaneous nerve fibers with a long and complete structure in the thin section4,5,6,7,8; comparatively, tissue clearing treatment on the large-sized sample can take a long time16,17,18,19. Considering their advantages and disadvantages, the present protocol is a proper choice for examining the detailed structure of the cutaneous nerve fibers within the transparently treated thick section, which is convenient to be observed and recorded under ordinary confocal microscopy. Utilizing this approach, one can observe longer cutaneous nerve fibers within the thick section compared to the thin section, and save experimental time for the tissue treatment compared to the large-sized samples with tissue clearing.
In this study, the cutaneous nerve fibers were labeled with CGRP. These kinds of nerve fibers belong to C and A&#948; sensory fibers, playing the role of transporting nociceptive signals and modulating vasodilatation12,13. Since CGRP-positive nerve fibers are located close to blood vessels and lymphatic vessels in the subcutaneous layer, it was also suggested to participate in wound healing by improving angiogenesis and lymphangiogenesis20,21. Similar to previous studies14,20,21,22, phalloidin was strongly expressed in the blood vessels with this triple-labeling experiment. In addition, LYVE1 is an integral membrane glycoprotein and is effectively employed as a biomarker for sorting rat dermal lymphatic endothelial cells15,23,24. Taking advantage of the triple fluorescent staining with CGRP, phalloidin, and LYVE1, together with the tissue clearing technique, a high-resolution image for better insight into the network of nerve fibers, blood vessels, and lymphatic vessels in the hairy and glabrous skin is presented.
It is to be noted that only the CGRP-positive nerve fibers were demonstrated in this study. Besides this kind of sensory nerve fiber, this protocol may also fit for examining other types of cutaneous nerve fibers with the corresponding antibodies. In addition, since phalloidin is highly expressed in the cytoskeletal component in smooth muscular and endothelial cells21,22,25, besides the blood vessels, the muscular and epidermal tissues were also labeled with phalloidin. However, according to the morphological characteristic, it is not difficult to identify blood vessels from the other kind of tissues.
In summary, the present protocol effectively explores the innervation of the hairy and glabrous skin on thicker sections by using a combination of immunofluorescence with clearing treatment. From the methodology perspective, it would be a benefit to investigate the other kinds of cutaneous nerve fibers and their spatial correlation with blood vessels and lymphatic vessels in the future.

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.

<table><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&amp;amp;L Alexa-Flour488</td><td>Abcam plc.</td><td>ab150105</td><td></td></tr><tr><td>Donkey anti-sheep IgG H&amp;amp;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&amp;amp;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 &amp;amp; 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 these tissue structures were further reconstructed in a 3D pattern, their distribution became easier to trace. It was shown that the CGRP-positive nerve fibers passed through the subcutaneous tissue and dermis to the epidermis. These nerve fibers ran in bundles in the subcutaneous tissue, branched within the dermis, and terminated in the epidermis (Figure 3). In contrast, phalloidin-positive blood vessels and LYVE1-positive lymphatic vessels are distributed in the subcutaneous tissue and dermis (Figure 3). Generally, the CGRP-positive nerve fibers ran parallel to or surrounded the blood vessels and lymphatic vessels, forming a 3D network in the hairy and glabrous skin.
With the conventional approach, although the nerve fibers, blood vessels, and lymphatic vessels were clearly labeled with CGRP, phalloidin, and LYVE1 in the thin sections, the observation of these structures is limited by the slice thickness and cannot be observed completely (Figure 4). The imaging technique rendered in-depth images for each object item from different positive fluorescent signals. After generating the image, the surface area of CGRP-positive nerve fibers was acquired through the Imaris software. In the section with a thickness of 30 &#181;m, there was no difference between hairy skin and glabrous skin in the surface area of CGRP-positive nerve fibers. In the 300 &#181;m thick tissue sections, compared with hairy skin, the surface area of CGRP-positive nerve fibers of glabrous skin was significantly increased (*P &#60; 0.05, Kruskal-Wallis nonparametric test, n = 3, Figure 5). Therefore, to compare the surface area of positive nerve fibers accurately, the 300 &#181;m cleared section is better than the traditional 30 &#181;m section. As a comparison, it is possible to acquire more opportunities for an ideal 3D image from the thick tissue section with the clearing treatment than in the conventional thin section.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63807/63807fig01.jpg" /> 
Figure 1: Photographs of experimental tools and key steps in the study. (A) Surgical tools (scalpel, shears, etc.). (B) The pump for perfusion. (C) Plantar and dorsal sides of hind paw after perfusion. (D) The sole and dorsum skins were removed from the hind paw. (E) Trimmed skin tissues. (F,G) Mounted skin tissues with 2% agarose at 37 &#176;C and 4 &#176;C. (H) Vibratory microtome. (I) Fixed skin tissues on the cutting supporter. (J) Set parameters for slicing. (K) Process of slicing. (L) Representative sections have a thickness of 300 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63807/63807fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63807/63807fig02.jpg" /> 
Figure 2: The fluorescent staining procedure and clearing of skin tissue. (A) A representation of the protocol steps involved in this study. (B) Outside views of skin tissue before and after the clearing treatment. <a href="https://www.jove.com/files/ftp_upload/63807/63807fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63807/63807fig03.jpg" /> 
Figure 3: Spatial correlation of nerve fibers, blood vessels, and lymphatic vessels following tissue clearing treatment on the thick skin section. (A,B) Representative images of the thick section of rat hindfoot&#39;s plantar and dorsal skin show the distribution of nerve fibers, blood vessels, and lymphatic vessels in the glabrous (A) and hairy (B) skin. (A1-A3) Panel A was separately shown with CGRP-labeling (A1), Pha-labeling (A2), and LYVE1-labeling (A3). (A4,A5; B1,B2) Panels (A) and (B) were adjusted in a 3D pattern and showed with the front (A4,B1) and back (A5,B2) views, respectively. Same scale bar for all panels. <a href="https://www.jove.com/files/ftp_upload/63807/63807fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63807/63807fig04.jpg" /> 
Figure 4: Spatial correlation of nerve fibers, blood vessels, and lymphatic vessels on the thin skin section with the conventional approach. (A,B) Representative images of the thick section of rat hindfoot&#39;s plantar and dorsal skin show the distribution of nerve fibers, blood vessels, and lymphatic vessels in the glabrous (A) and hairy (B) skin. (A1,A2; B1,B2): Panels (A) and (B) were adjusted in a 3D pattern and showed with the front (A1,B1) and back (A2,B2) views, respectively. Same scale bar for all panels. <a href="https://www.jove.com/files/ftp_upload/63807/63807fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63807/63807fig05.jpg" /> 
Figure 5: A histogram showing the surface area of CGRP-positive nerve fibers in hairy and glabrous skin. (A) In the section with a thickness of 30 &#181;m, there was no difference between hairy skin and glabrous skin in the surface area of CGRP-positive nerve fibers. (B) In the section with a thickness of 300 &#181;m, compared with hairy skin, the surface area of CGRP-positive nerve fibers of glabrous skin was significantly increased (*P &#60; 0.05, n = 3). <a href="https://www.jove.com/files/ftp_upload/63807/63807fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

demonstrating-hairy-glabrous-skin-innervation-3d-pattern-using

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.5K Views.  China Academy of Chinese Medical Sciences. 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 µm tissue sections under confocal microscopy.

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

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

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

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

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

Medicine

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Biology

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

Efficient 3D Skin Models for Flexible Research Application

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical industries. In the European Union, there has been a total ban on testing cosmetic products and their ingredients on animals. In the case of medicine and pharmaceuticals, this possibility is also constantly limited. In accordance with the 3Rs principle, it is becoming more and more common to test individual compounds as well as entire formulations on artificially created models. The cheapest and most widely used are the 2D models, which consist of a cell monolayer but do not reflect the real interactions between the cells in the tissue. Although the commercially available 3D models provide a better representation of the tissue, they are not used on a large scale. This is because they are expensive, the waiting time is quite long, and the available models are frequently limited to only those typically used. 
In order to move the conducted research to a higher level, we have optimized the procedures of various 3D skin model preparations. The described procedures are cheap and simple to prepare as they can be applied in numerous laboratories and by researchers with different experiences in cell culture.

Author Spotlight: Enhancing Skin Model Diversity with Cost-Effective 3D Cellular Models

Here, we present inexpensive and simple procedures to introduce various 3D skin models for routine research in a cell culture laboratory. Researchers can create models tailored to their needs without relying on commercially available models.

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical ...

Bioengineering

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

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

Summary

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Chapters in this video

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

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Cutaneous Surgical Denervation: A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations