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.
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.
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 µm 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.
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
- Inject 250 mg/kg of tribromoethanol solution (see Table of Materials) intraperitoneally into the rat to induce euthanasia.
- 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).
- After perfusion, use a scalpel to remove the skin of the dorsum and sole from the hind paw.
- 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
- 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.
- Prepare 2% agarose in 1x PBS by heating in a micro-oven for 2 min until the agarose dissolves (see Table of Materials).
- After washing, embed the skin tissues into 2% agarose at 37 °C, and place them inside the icebox for cooling (Figure 1F,G).
- 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).
- After slicing, remove the agarose from the sections, and store them in a six-well plate with 1x PBS (pH 7.4) (Figure 1L).
- 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.
- 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).
- 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.
- 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).
- 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.
- 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.
- 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).
- 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.
- Slice the tissue sections at 30 µm on a microtome in the transversal direction and mount them on the slide.
- 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.
- 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.
- 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.
- 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
- Observe the stained samples under a fluorescent microscope, and then take the images using a confocal microscope.
- 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).
- 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.
- 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.
- 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).
- Demonstrate the images in the pattern of 3D reconstruction with the image processing system.
- 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.
- Acquire data in the surface area of positive nerve fibers with the cell imaging software. Select the rendered image | Statistics | Detailed | Specific Values | Volume.
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 µm, there was no difference between hairy skin and glabrous skin in the surface area of CGRP-positive nerve fibers. In the 300 µm thick tissue sections, compared with hairy skin, the surface area of CGRP-positive nerve fibers of glabrous skin was significantly increased (*P < 0.05, Kruskal-Wallis nonparametric test, n = 3, Figure 5). Therefore, to compare the surface area of positive nerve fibers accurately, the 300 µm cleared section is better than the traditional 30 µm 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.
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 °C and 4 °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 µm. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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'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. Please click here to view a larger version of this figure.
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'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. Please click here to view a larger version of this figure.
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 µm, 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 µm, compared with hairy skin, the surface area of CGRP-positive nerve fibers of glabrous skin was significantly increased (*P < 0.05, n = 3). Please click here to view a larger version of this figure.
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 Aδ 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 authors have nothing to disclose.
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).
|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|
|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|
|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|
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