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 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 problem^9,10. 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 skin^3,4,7. To trace the cutaneous nerve fibers at a longer distance, the skin tissue was sliced at the thickness of 300 µm for immunohistochemical and histochemical staining, followed by tissue clearing treatment. CGRP was used to label the sensory nerve fibers^12,13. 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, respectively^14,15.

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.

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 250 mg/kg of tribromoethanol solution (see Table of Materials) intraperitoneally into the rat to induce euthanasia.
2. 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).
3. After perfusion, use a scalpel to remove the skin of the dorsum and sole from the hind paw.
   1. 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 °C
   2. 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 °C (Figure 1C–E).

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 µm following tissue clearing treatment.

1. Prepare 2% agarose in 1x PBS by heating in a micro-oven for 2 min until the agarose dissolves (see Table of Materials).
2. After washing, embed the skin tissues into 2% agarose at 37 °C, and place them inside the icebox for cooling (Figure 1F,G).
3. Fix the mounted tissue on the vibratory microtome with the ice water and slice them at a thickness of 500 µm 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).
4. After slicing, remove the agarose from the sections, and store them in a six-well plate with 1x PBS (pH 7.4) (Figure 1L).
5. Incubate the tissue sections in a solution of 2% Triton X-100 in 1x PBS (TriX-PB) overnight at 4 °C. See Figure 2 for the following procedure.
6. Place the tissue sections into the blocking buffer and rotate at 72 rpm on the shaker overnight at 4 °C.
   NOTE: The blocking buffer composition is 10% normal donkey serum, 1% Triton-X 100, and 0.2% sodium azide in 1x PBS (see Table of Materials).
7. 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 °C.
   NOTE: The dilution buffer composition is 1% normal donkey serum, 0.2% Triton-X 100, and 0.2% sodium azide in 1x PBS.
8. Wash the tissue sections twice with washing buffer at room temperature, then keep them on the shaker overnight at 4 °C in washing buffer.
   NOTE: The washing buffer composition is 0.2% Triton-X 100 in 0.1 M PB (pH 7.4).
9. The next day, transfer the tissue sections into the mixed solution containing the secondary antibodies of donkey anti-mouse IgG H&L Alexa-Flour488 (1:500) and donkey anti-sheep IgG H&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 °C.
10. 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 °C.
11. 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).
12. Mount the cleared tissues on the slide, circle the tissues with a spacer, and stick the gap with fresh tissue clearing reagent and coverslip.

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 µm following conventional techniques.

1. Slice the tissue sections at 30 µm on a microtome in the transversal direction and mount them on the slide.
2. 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.
3. 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 °C.
4. 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&L Alexa-Flour488 (1:500) and donkey anti-sheep IgG H&L Alexa-Flour405 (1:500), as well as Alexa-Flour594 phalloidin (1:1000) on the sections and incubate for 1 h at room temperature.
5. Before microscopic observation, wash the sections in 0.1 M PB (pH 7.4) three times, then cover them with coverslips in 50% glycerin.

4. Imaging and analyses

1. Observe the stained samples under a fluorescent microscope, and then take the images using a confocal microscope.
2. Capture 30 images (Z-stacks), each in 10 µm frames, from each 300 µm thick section and integrate a single in-focus image using an image processor of the confocal microscopy system (see Table of Materials).
   1. 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 µm is the diameter of the confocal pinhole (10x objective lens, NA: 0.4). The image capture resolution was 1024 × 1024 pixels.
3. For the conventional samples (step 3), capture 30 images (Z-stacks) in 1 µm frames from each 30 µm thick section and further treat these images as mentioned above (steps 4.1-4.2).
4. Demonstrate the images in the pattern of 3D reconstruction with the image processing system.
5. 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.
6. Acquire data in the surface area of positive nerve fibers with the cell imaging software. Select the rendered image | Statistics | Detailed | Specific Values | Volume.

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