Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Summary

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Abstract

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Introduction

The skin is one of the largest organs in the body, with important functions in somato-sensation, insulation/thermoregulation, and immune defense¹. Understanding the molecular and cellular basis of skin development and function has been of longstanding interest because of the fundamental importance of skin as a biological system and its relevance to dermatology. Mammalian skin contains a variety of multicellular structures, including stratified layers of keratinocytes, dermal connective tissue, several types of hair follicles, sebaceous glands, arrector pili muscles, blood vessels, and at least a dozen distinct classes of afferent (sensory) and efferent nerv....

Protocol

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocol MO11M29 of the Johns Hopkins Medical Institutions. Consult your local Institutional Animal Care and Use Committee guidelines for approved methods of euthanasia. Wear gloves, lab coat, and safety glasses when handling aldehyde fixatives or organic solvents.

1. Preparation of Materials

1. Pouring Sylgard Plates
   1. Follow the manufacturer's instru....

Results

Brightfield imaging of skin flatmounts can be used to image cutaneous sensory afferents (Figure 3A¹⁰) and hair follicle patterns based on melanin pigmentation (Figure 4). Confocal imaging of skin flatmounts can be used to define the geometry of (1) Merkel cell clusters, visualized with anti-cytokeratin-6 or with AM dye uptake (Figures 3I-L), (2) arrector pili muscles, visualized with anti-smooth muscle actin (Figures 3G,H), (3) sebaceous gland.......

Discussion

Mastery of the dissection methods described above requires only patience, a steady hand, and a few good dissection tools. The dorsal skin dissection is relatively easy, but the tail and foot skin dissections – especially at early postnatal ages – are more challenging. At early prenatal ages (e.g., before E15), the skin is difficult to remove without tearing it. Conveniently, for many studies of growth and patterning of skin structures in mice, the events of interest occur postnatally, as seen for exa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-bromo-4-chloro-indolyl phosphate (BCIP)	Roche	11383221001
AM1-43	Biotium	70024
AM4-65	Biotium	70039
Benzyl alcohol	Sigma	402834
Benzyl benzoate	Sigma	B-6630
Confocal microscope	Zeiss	LSM700
Cy3-alpha smooth muscle actin antibody	Sigma	C6198	1:400
Cytokeratin-8	Developmental Studies Hybridoma Bank	TROMA-I-c	1:500
Dissecting microscope
Dissection tools	Fine Science Tools		scissors and forceps
Electric razor
Fluoromount G	EM Sciences	17984-25
Formalin	Sigma	HT501320
Glass dishes	Pyrex		6 cm and 10 cm diameter
Glass plates	Amersham Biosciences	SE202P-10	10 cm x 8 cm x 1 mm
Hair remover	Nair
Horizontal rotating platform	Hoefer	PR250 Orbital shaker
Insect pins	Fine Science Tools	26002-20
Ketamine/xylazine	Sigma	K113
Nitroblue tetrazolium (NBT)	Roche	11383213001
Oil Red O	Sigma	O0625
Paraformaldehyde	Sigma	P6148
Razor Blades	VWR	55411-055
Secondary antibodies	Invitrogen		Alexa-dye conjugated
Sylgard-184	Fisher Scientific	NC9020938
Tissue culture plastic dishes			10 cm diameter
Tissue culture plates			6- and 12-well