CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

Summary

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

Abstract

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

Introduction

The skin is essential for our survival. It consists of three main layers the outer epidermis, the dermis and the hypodermis. The epidermis is a highly regenerative tissue. It is a squamous stratified epithelium, consisting mostly of keratinocytes. Keratinocytes are born in the basal layer, and move upwards through the suprabasal layers while differentiating, and eventually they are shed in the outer cornified layer about a month after their birth. The epidermis develops a number of appendages including the hair follicles and sebaceous glands. The hair follicles also regenerate in a cyclical fashion throughout life¹. The regenerative capacity of the epidermi....

Protocol

Ethics Statement: All procedures involving animal subjects follow the guidelines of the Animal Care and Ethics Committee (ACEC) at UNSW Australia under approved ACEC protocol 13/64B.

1. Preparation of the Transparent Mouse Skin Tissue

Note: All mice used in this study were on a C57BL/6 genetic background

1. Collection of mouse skin tissue.
   1. Humanely euthanize the mice by cervical dislocation.
   2. Carefully remove hairs from the relevant skin area with a trimmer taking care not to wound the skin.
   3. Wash skin to decontaminate with 70% ethanol in Phosphate Buffered Saline (PBS).

Results

Full thickness dorsal skin biopsies of adult wildtype mice were clarified, stained with an antibody binding basal keratinocyte marker Keratin14 (K14), and nuclei were counterstained with DAPI staining solution (Figure 2 and Movie 1).

DAPI-positive nuclei were visible throughout the sample (Figure 2A, C), and K14 staining was visible exclusively in the one-cell thick basal layer of the interfollicular epidermis, and outlining the sebaceous glan.......

Discussion

The regulatory mechanisms controlling skin development and homeostasis are most commonly studied in 2D using tissue sectioning and histological staining or labeling with antibodies, which enables only a restricted appreciation of skin morphology, cell populations or protein expression. A number of methods have been developed to improve visualization of the spatial organization of cells and proteins at single cell resolution in 3 dimensions in epidermal whole mounts^10-13. Some of these however involve separatio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Paraformaldehyde	Sigma-Aldrich	P6418
Ethanol 96% (undenaturated)	Chem-supply	UN1170
Nile Red	Sigma-Aldrich	72485-100MG
4’,6-diamidino-2-phenylindole (DAPI)	Roche	10236276001
N,N,N’,N’-tetrakis (2-hydroxypropyl) ethylenediamine	Merck Millipore	821940
Polyethylene glycol mono-p-isooctylphenyl ether	Merck Millipore	648462
Triton X-100	Merck Millipore	648462
Sucrose	Sigma-Aldrich	S0389
Optimal Cutting Temperature (OCT) Compound	Tissue-Tek	4583
anti-Keratin14 antibody	Covance	PRB-155P
anti-Ki67 antibody	Abcam	ab16667
Donkey anti-rabbit Alexa 594	Life Technologies	A21207
Dimethylsulfoxide	Sigma-Aldrich	D2650
Urea	Merck Millipore	66612
2,2′,2′’-nitrilotriethanol	Merck Millipore	137002
Confocal Microscope	Nikon Instruments Inc	Nikon A1 - Confocal Microscope
cruZer6 Face Trimmer	Braun	Braun cruZer6 Face
Sodium azide	Sigma-Aldrich	438456