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

Summary

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

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

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 components^1,....

Protocol

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.
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Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 x Phosphate Buffered Saline	KD Medical	RGE-3210	PBS, without Ca2+/Mg2+
Hank’s Balanced Salt Solution	Gibco	14025-092	HBSS, with Ca2+/Mg2+
16% Paraformaldehyde	Electron Microscopy Sciences	15710	PFA, fixative, diluted in PBS
Triton X-100	Sigma	X100	Detergent
Normal goat serum	Gibco	16210064	Component of blocking/washing buffer
Normal donkey serum	Jackson Immuno research	017-000-121	Component of blocking/washing buffer
Curved fine tweezers	Dumont	RS-5047
Curved tweezers	Integra Miltex Vantage	V918-782, V918-784
Filter Unit 0.45 mm	Thermo Scientific	157-0045	For filtration
1 mL syringe	Coviden	8881501400	For filtration
Syringe filter Unit 0.22 mm	Millex-GV	SLGVR04NL	For filtration
ProLong Gold	Thermo Scientific	P36934	Anti-fade mounting medium
Nail Polish	Electron Microscopy Sciences	72180	For sealing
Dissecting microscope	Leica	MZ95
Confocal microscope	Leica	TCS SP5
Photoshop CC 2017	Adobe		Graphics editor software
Illustrator CC 2017	Adobe		Graphics editor software
Image J	NIH		Image processing software
Anti-PECAM-1 (CD31) antibody	Millipore	MAB1398Z	Hamster IgG, vascular endothelial cell marker, 1:300
Anti-PECAM-1 (CD31) antibody	BD Pharmingen	553369	Rat IgG2a kappa, vascular endothelial cell marker, 1:300
Anti-αSMA antibody conjugated with cy-3	Sigma	C6198	Mouse IgG2a, vascular smooth muscle cell marker, 1:500
Anti-EphB1 antibody	Santa Cruz	sc-9319	Goat polyclonal, venous endothelial cell marker, 1:100
Anti-neuron-specific Class III β-tubulin (Tuj1)	Abcam	AB18207	Tuj1, Rabbit polyclonal IgG, pan-axonal marker, 1:500
Anti-Tuj1 antibody	Covance	MMS-435P	Mouse IgG2a, pan-axonal marker, 1:500
Anti-MBP antibody	Abcam	AB40390	Rabbit polyclonal IgG, myelination marker, 1:200
Anti-Tyrosine Hydroxylase antibody	Chemicon	AB152	Rabbit polyclonal, sympathetic neuron marker, 1:500
Anti-Peripherin antibody	Chemicon	AB1530	Rabbit polyclonal, peripheral neuron marker, 1:1000
Anti-CD11b antibody	Bio-Rad	MCA74G	Rat IgG2b, inflammatory cell marker (macrophages), 1:50
Anti-CD45 antibody	Thermo Fisher Scientific	14-0451-85	Rat IgG2b kappa, pan-hematopoietic cell marker, 1:500
Anti-CD3 antibody	Bio-Rad	MCA1477T	Rat IgG1, immune cell marker, 1:100
Anti-CD45R (B220) antibody	Thermo Fisher Scientific	14-0452	Rat IgG2a kappa, inflammatory cell marker, 1:200
Anti-GFP antibody	Thermo Fisher Scientific	A11122	Rabbit polyclonal, 1:300
Anti-GFP antibody	Abcam	Ab13970	Chicken polyclonal, 1:500
Anti-β-gal antibody	Cappel	55976	Rabbit polyclonal, 1:5000
Anti-RFP antibody	Abcam	Ab62341	Rabbit polyclonal, 1:300
Goat anti-rabbit IgG (H+L) Alexa 488	Thermo Fisher Scientific	A11034	Rabbit polyclonal secondary antibody, 1:250
Goat anti-hamster IgG (H+L) Alexa 647	Jackson Immuno research	127-605-160	Hamster polyclonal secondary antibody, 1:250
Goat anti-rat IgG (H+L) Alexa 594	Jackson Immuno research	112-585-167	Rat polyclonal secondary antibody, 1:250
Goat anti-mouse IgG2a Alexa 633	Thermo Fisher Scientific	A21136	Mouse IgG2a secondary antibody, 1:250