Method Article

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

DOI:

10.3791/57406

⸱

March 29th, 2018

 ,  , 
1Laboratory of Stem Cell and Neuro-Vascular Biology, Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, 2Earle A. Chiles Research Institute, Robert W. Franz Cancer Center, Providence Portland Medical Center

In This Article

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

Introduction

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Skin is comprised of three layers: the epidermis, dermis and hypodermis. It has been used as a model system to study stem cell maintenance, differentiation, and morphogenesis in development as well as the regeneration, tumorigenesis, and inflammation in adult. Skin is richly vascularized and innervated such that the development of the peripheral nervous system and vascular system is well-coordinated.

We have previously demonstrated a whole-mount embryonic skin imaging technique with multiple labeling to study intact peripheral nerves and blood vessels including their cellular components1,....

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

All experiments in this section were performed under approval from the National Heart, Lung, and Blood Institute (NHLBI) Animal Care and Use Committee.

1. Adult Mouse Ear Skin Collection

  1. Euthanize adult mice by carbon dioxide (CO2) exposure in a closed chamber and then confirm the euthanasia by cervical dislocation.
    NOTE: The experiment follows the National Institutes of Health (NIH) guideline for the euthanasia method.
  2. Dissect the ear from the base and place it in a 35 x 10 mm2 Petri dish containing 2 mL of Hank's Balanced Salt Solution (HBSS). Briefly trim hairs with scissors.
    3. <....

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Adult mouse posterior ear skin (Figure 1A) and anterior ear skin (Figure 1B) were immunostained with antibodies to αSMA (red), Tuj1 (green), and PECAM-1 (blue). Posterior skin was immunostained to study neuro-immune distribution using antibodies to CD11b (red) and MBP (green), together with Tuj1 (blue) (Figure 2A). Distribution of CD11b+ inflammatory cells, including macrophages was detect.......

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

This protocol describes the whole-mount immunonohistochemical imaging of adult ear skin for the analysis of neuro-vascular structures and immune cell distribution. We believe this method has numerous experimental advantages for researchers to study branching morphogenesis and the patterning of peripheral nerves and blood vessels, as well as three-dimensional distribution of skin components including immune cells and hair follicles. The results of the imaging can be quantified using imaging softwares for further quantitat.......

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have nothing to disclose.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

We thank K. Gill for the laboratory management and technical support, J. Hawkins and the staff of National Institutes of Health (NIH) Building 50 animal facility for the assistance with mouse care, and R. Reed and F. Baldrey for administrative assistance. Thanks also to S. Motegi and M. Udey for sharing their ear skin dissection protocol, N. Burns for editorial help, and members of the Laboratory of Stem Cell and Neuro-Vascular Biology for technical help and thoughtful discussion. T. Yamazaki was supported by the Japan Society for the Promotion of Science (JSPS) NIH-KAITOKU. This work was supported by the Intramural Research Program of the National Heart, Lung, and Bl....

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
10 x Phosphate Buffered SalineKD MedicalRGE-3210PBS, without Ca2+/Mg2+
Hank’s Balanced Salt SolutionGibco14025-092HBSS, with Ca2+/Mg2+
16% ParaformaldehydeElectron Microscopy Sciences15710PFA, fixative, diluted in PBS
Triton X-100SigmaX100Detergent
Normal goat serumGibco16210064Component of blocking/washing buffer
Normal donkey serumJackson Immuno research017-000-121Component of blocking/washing buffer
Curved fine tweezersDumontRS-5047
Curved tweezersIntegra Miltex VantageV918-782, V918-784
Filter Unit 0.45 mmThermo Scientific157-0045For filtration
1 mL syringeCoviden8881501400For filtration
Syringe filter Unit 0.22 mmMillex-GVSLGVR04NLFor filtration
ProLong GoldThermo ScientificP36934Anti-fade mounting medium
Nail PolishElectron Microscopy Sciences72180For sealing
Dissecting microscopeLeicaMZ95
Confocal microscopeLeicaTCS SP5
Photoshop CC 2017AdobeGraphics editor software
Illustrator CC 2017AdobeGraphics editor software
Image JNIHImage processing software
Anti-PECAM-1 (CD31) antibodyMilliporeMAB1398ZHamster IgG, vascular endothelial cell marker, 1:300
Anti-PECAM-1 (CD31) antibodyBD Pharmingen553369Rat IgG2a kappa, vascular endothelial cell marker, 1:300
Anti-αSMA antibody conjugated with cy-3SigmaC6198Mouse IgG2a, vascular smooth muscle cell marker, 1:500
Anti-EphB1 antibodySanta Cruzsc-9319Goat polyclonal, venous endothelial cell marker, 1:100
Anti-neuron-specific Class III β-tubulin (Tuj1)AbcamAB18207Tuj1, Rabbit polyclonal IgG, pan-axonal marker, 1:500
Anti-Tuj1 antibodyCovanceMMS-435PMouse IgG2a, pan-axonal marker, 1:500
Anti-MBP antibodyAbcamAB40390Rabbit polyclonal IgG, myelination marker, 1:200
Anti-Tyrosine Hydroxylase antibodyChemiconAB152Rabbit polyclonal, sympathetic neuron marker, 1:500
Anti-Peripherin antibodyChemiconAB1530Rabbit polyclonal, peripheral neuron marker, 1:1000
Anti-CD11b antibodyBio-RadMCA74GRat IgG2b, inflammatory cell marker (macrophages), 1:50
Anti-CD45 antibodyThermo Fisher Scientific14-0451-85Rat IgG2b kappa, pan-hematopoietic cell marker, 1:500
Anti-CD3 antibodyBio-RadMCA1477TRat IgG1, immune cell marker, 1:100
Anti-CD45R (B220) antibodyThermo Fisher Scientific14-0452Rat IgG2a kappa, inflammatory cell marker, 1:200
Anti-GFP antibodyThermo Fisher ScientificA11122Rabbit polyclonal, 1:300
Anti-GFP antibodyAbcamAb13970Chicken polyclonal, 1:500
Anti-β-gal antibodyCappel55976Rabbit polyclonal, 1:5000
Anti-RFP antibodyAbcamAb62341Rabbit polyclonal, 1:300
Goat anti-rabbit IgG (H+L) Alexa 488Thermo Fisher ScientificA11034Rabbit polyclonal secondary antibody, 1:250
Goat anti-hamster IgG (H+L) Alexa 647Jackson Immuno research127-605-160Hamster polyclonal secondary antibody, 1:250
Goat anti-rat IgG (H+L) Alexa 594Jackson Immuno research112-585-167Rat polyclonal secondary antibody, 1:250
Goat anti-mouse IgG2a Alexa 633Thermo Fisher ScientificA21136Mouse IgG2a secondary antibody, 1:250

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin. Cell. 109, 693-705 (2002).
  2. Mukouyama, Y. S., James, J., Nam, J., Uchida, Y.

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Whole mount Confocal MicroscopyAdult Ear SkinNeuro vascular BranchingImmune Cell DistributionPeripheral NervesBlood VesselsImmunohistochemical StainingFluorescent SignalsThree dimensional ArchitectureMouse Model

Related Articles