JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

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

Published: August 4th, 2016

DOI:

10.3791/54401

1The School of Medical Sciences, University of New South Wales Australia, 2Neuroscience Research Australia, 3Biomedical Imaging Facility, University of New South Wales Australia
* These authors contributed equally

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.

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.

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....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 sk.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 mounts10-13. Some of these however involve separatio.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

We thank Australian Bio-Resources (Garvan Institute, Australia), the Biological Resources Centre (UNSW Australia) and the Animal Care & Ethics Committee for support with animal experimentation. This work was supported by the National Health and Medical Research Council of Australia (Project Grant APP1062720). Dr. Cesar P. Canales is recipients of a CONICYT-Becas Chile scholarship (#72101076). Mr. Bassem Akladios is a recipient of University International Postgraduate Award by UNSW Australia.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

  1. Fuchs, E. Scratching the surface of skin development. Nature. 445 (7130), 834-842 (2007).
  2. Watt, F. M., Lo Celso, C., Silva-Vargas, V. Epidermal stem cells: an update. Curr Opin Genet Dev. 16 (5), 518-524 (2006).
  3. Hardy, M. H. The secret life of the hair follicle. Trends Genet. 8 (2), 55-61 (1992).
  4. Lim, X., Nusse, R. Wnt signaling in skin development, homeostasis, and disease. Cold Spring Harb Perspect Biol. 5 (2), (2013).
  5. Susaki, E. A., et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell. 157 (3), 726-739 (2014).
  6. Susaki, E. A., Tainaka, K., Perrin, D., Yukinaga, H., Kuno, A., Ueda, H. R. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc. 10 (11), 1709-1727 (2015).
  7. Tainaka, K., et al. Whole-body imaging with single-cell resolution by tissue decolorization. Cell. 159 (4), 911-924 (2014).
  8. Beverdam, A., Claxton, C., Zhang, X., James, G., Harvey, K. F., Key, B. Yap controls stem/progenitor cell proliferation in the mouse postnatal epidermis. J Invest Dermatol. 133 (6), 1497-1505 (2013).
  9. Fuchs, E., Green, H. Changes in keratin gene expression during terminal differentiation of the keratinocyte. Cell. 19 (4), 1033-1042 (1980).
  10. Braun, K. M., Niemann, C., Jensen, U. B., Sundberg, J. P., Silva-Vargas, V., Watt, F. M. Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis. Development. 130 (21), 5241-5255 (2003).
  11. Hamilton, E., Potten, C. S. Influence of hair plucking on the turnover time of the epidermal basal layer. Cell Tissue Kinet. 5 (6), 505-517 (1972).
  12. Morris, R. J., Fischer, S. M., Slaga, T. J. Evidence that a slowly cycling subpopulation of adult murine epidermal cells retains carcinogen. Cancer Res. 46 (6), 3061-3066 (1986).
  13. Schweizer, J., Marks, F. A developmental study of the distribution and frequency of Langerhans cells in relation to formation of patterning in mouse tail epidermis. J Invest Dermatol. 69 (2), 198-204 (1977).
  14. Chang, H., Wang, Y., Wu, H., Nathans, J. Flat mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology. J Vis Exp. (88), e51749 (2014).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved