Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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 µm 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.

Introduction

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 ....

Protocol

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 ± 15 g) were used in this study. All animals were housed in a 12 h light/dark cycle with controlled temperature and hu.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
1x phosphate-buffered salineSolarbio Life SciencesP1020pH 7.2-7.4, 0.01 Mol
2,2,2-TribromoethanolSigma Life ScienceT48402-5G
Confocal fluorescence microscopyOlympus CorporationFluoview FV1200
Donkey anti-mouse IgG H&L Alexa-Flour488Abcam plc.ab150105
Donkey anti-sheep IgG H&L Alexa-Flour405Abcam plc.ab175676
EP tubeWuxi NEST Biotechnology Co.6150011.5 mL
Freezing stage sliding microtome systemLeica BiosystemsCM1860
Imaris SoftwareOxford Instrumentsv.9.0.1
IRIS standard scissorWPI (World Precision Instruments Inc.)503242
iSpacerSunJin Lab co.IS005
Micro forceps-StrRWDF11020-11
Mouse monoclonal anti-CGRP antibodySanta cruz biotechnology, Inc.sc-57053
Neutral buffered FormalinSolarbio Life SciencesG216110%
Normal donkey serumJackson ImmunoResearch Laboratories017-000-1210 mL
Peristaltic pumpLonger Precision Pump Co., LtdBT300-2J
Phalloidin Alexa-Fluor 594Thermo Fisher ScientificA12381
RapiClear 1.52 solutionSunJin Lab co.RC15200110 mL
Regular agaroseGene Company LimitedG-10
SEMKEN 1 x 2 Teeth Tissue Forceps-StrRWDF13038-12
Sheep polyclonal anti-LYVE1 antibodyR&D Systems, Inc.AF7939
Six-well plateCorning Incorporated3335
Sodium azideSigma Life ScienceS200225 g
SucroseSigma Life ScienceV900116500 g
Super GlueHenkel AG & Co.Pattex 502
Surgical HandlesRWDS32003-12
Triton X-100Solarbio Life Sciences9002-93-1100 mL
UrethaneSigma Life ScienceU2500500 g
VANNAS spring scissorsRWDS1014-12
Vibratory microtomeLeica BiosystemsVT1200S

References

  1. Vidal Yucha, S. E., Tamamoto, K. A., Kaplan, D. L. The importance of the neuro-immuno-cutaneous system on human skin equivalent design. Cell Proliferation. 52 (6), 12677 (2019).
  2. Wu, H., Williams, J., Nathans, J.

Explore More Articles

Tissue ClearingFluorescent StainingSkin InnervationCGRPLYVE 13D ImagingConfocal MicroscopyTissue SectioningTissue EmbeddingTissue Clearing Reagent

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved