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

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 fibers^1,2,3. Although skin innervation has been widely studied previously with various histological methods, such as staining on whole-mount skin and tissue sections^4,5,6, the detailed effective demonstration of cutaneous nerve fibers is still a challenge^7,8. 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 humidity and allowed free access to food and water.

1. Perfusion and sample preparation

1. Inject ....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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