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 epidermis is enabled by the presence of stem and progenitor cells that are located in the basal layer of the interfollicular epidermis and hair follicle2.
Many signaling pathways have been implicated in epidermal development and regeneration. Some of these occur within the epidermis only, such as the Hedgehog pathway. Other signaling events take place between dermis and epidermis3. For instance, Wnt signals from the dermis are thought to be important for hair follicle development, and they are secreted by the dermal papilla at the onset of anagen to activate hair follicle bulge stem/progenitor cell proliferation and hair follicle growth4. It is important to understand the cellular and molecular mechanisms that control epidermal development and regeneration to better understand how they may be perturbed in regenerative skin disease such as skin cancer.
This article describes a Clear, Unobstructed Brain Imaging cocktails and Computational analysis (CUBIC) protocol5-7 to clarify whole mount skin preparations, and visualize protein expression patterns in 3 dimensions at single cell resolution by confocal microscopy. The CUBIC method involves immersion of skin tissue in two aminoalcohol-based chemical cocktails. These solutions adjust the refractive indices in the skin sample, leaving the tissue transparent and the proteins intact, allowing immunodetection at single cell resolution.
Using this CUBIC protocol, the basal and proliferating keratinocyte populations in the interfollicular epidermis and in the hair follicle were imaged in full thickness skin biopsies of wildtype mice using anti-Keratin14 (K14) and anti-Ki67 antibodies. Sebaceous glands in wildtype skin biopsies were also visualized using Nile Red staining. Lastly, the basal keratinocyte populations in wildtype and hyperplastic YAP2-5SA-ΔC skin biopsies were compared8.
This CUBIC protocol enables visual assessment of protein expression in full thickness skin biopsies at single cell resolution, and is an important tool to appreciate epidermal anatomy and morphological defects in the skin of genetically modified mice, and to investigate the cellular and molecular mechanisms underlying epidermal development and regeneration.
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
2. Immunofluorescence Staining
3. Nile Red Staining
4. Imaging
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 glands (black asterisks), the outer root sheaths of hair follicles, and in the secondary hair germs (Figure 2B, C), as previously published9. K14 staining intensity was high in the interfollicular epidermis, the distal hair follicle and the secondary hair germs (white asterisks), and it was relatively low in the bulge area of the hair follicle (Bu in Figure 2C). The dermal papillae were also clearly visible through DAPI labeling (arrow in Figure 2C).
To visualize proliferating cells, full thickness dorsal skin biopsies in telogen of adult wildtype mice were clarified and stained with an anti-Ki67 antibody, and nuclei were counterstained with DAPI solution (Figure 3, and Movie 2).
Proliferating keratinocytes were detected in the basal interfollicular epidermis (IFE in Figure 3C), and in the isthmus (Is in Figure 3C) of the hair follicles, but not in the bulge region (Bu in Figure 3C).
To visualize sebaceous glands, full thickness dorsal skin biopsies in anagen of adult wildtype mice were clarified and stained with Nile Red that labels the fat content of sebocytes, and nuclei were counterstained with DAPI staining solution (Figure 4 and Movie 3). Nile Red-positive sebaceous glands were observed in the isthmus region of the hair follicles (SB in Figure 4C), showing that the CUBIC protocol does not significantly affect the morphology of these fat-containing structures. Unexpectedly, hair shafts were also labeled with Nile Red (HS in Figure 4C).
To visualize the morphological changes in the epidermis of YAP2-5SA-ΔC transgenic mice expressing a hyperactive YAP mutant protein in basal keratinocytes8, full thickness dorsal skin biopsies of adult wildtype and YAP2-5SA-ΔC transgenic littermate mice were clarified and stained with the anti-K14 antibody, and nuclei were counterstained with DAPI solution (Figure 5, and Movie 4 (wildtype) and Movie 5 (YAP2-5SA-ΔC)).
In the YAP2-5SA-ΔC transgenic skin, the hyperplasia of the interfollicular epidermis (top double arrows in Figure 4F) was clearly visible, and the hair follicles were longer and displayed the K14-labeled cell masses proximally in the abnormal hair follicles (lower double arrows in Figure 5F), representing the enlarged stem cell populations, as previously described8.
Figure 1: Preparation of the Imaging Chamber. A: Consumables required for the preparation of the imaging chamber are blue tack, play dough or similar, and 2 coverslips (24 x 50 mm). B: prepare two thin strips of blue tack (diameter approximately 2 mm x 2 cm), and two coverslips. C: Place blue tack strips on one cover slip, allowing enough space for the skin biopsy. Place the skin biopsy in a drop of CUBIC2 solution in between the two strips. D: Place second coverslip on blue tack strips to cover the skin biopsy. Please click here to view a larger version of this figure.
Figure 2: K14/DAPI Labeling of Dorsal Skin in Telogen of Adult Wildtype Mice. 3D volume reconstruction of a dorsal skin biopsy in telogen of an adult wildtype mouse labeled with a K14 antibody (Red in B, C), and DAPI nuclear counterstain (blue in A, C). K14 staining is strong in the interfollicular epidermis (IFE), the isthmus and the sebaceous glands (black asterisks), and the secondary hair germs (white asterisk), and relatively low in the bulge area (Bu). Scale bars 50 µm. Please click here to view a larger version of this figure.
Figure 3: Ki67 Labeling of Dorsal Skin in Telogen of Adult Wildtype Mice. 3D volume reconstructions of a dorsal skin biopsy of an adult wildtype mouse labeled with a Ki67 antibody (Red in B, C), and DAPI nuclear counterstain (blue in A, C). Proliferating Ki67-positive keratinocytes are evident in the basal interfollicular epidermis (IFE), and in the isthmus region (Is), but not in the bulge (Bu) of the telogen hair follicle. Scale bars 50 µm. Please click here to view a larger version of this figure.
Figure 4: Nile Red Staining of Dorsal Skin in Anagen of Adult Wildtype Mice. 3D volume reconstruction of a dorsal skin biopsy in anagen of an adult wildtype mouse labeled with Nile Red (red in B, C), and DAPI nuclear counterstain (blue in A, C). Nile Red staining is visible in the sebocytes (SB) and in the hair shaft (HS). Scale bars 50 µm. Please click here to view a larger version of this figure.
Figure 5: Morphological Abnormalities in the YAP2-5SA-ΔC Epidermis. 3D volume reconstructions of dorsal skin biopsies of adult wildtype (A–C) and YAP2-5SA-ΔC transgenic littermate mice (D–F) labeled with a K14 antibody (red in B, C, E, F), and DAPI nuclear counterstain (blue in A, C, D, F). The epidermal hyperplasia of the YAP2-5SA-ΔC epidermis is apparent in the interfollicular epidermis (top double arrows in F) and in the hair follicles, which display the characteristic stem/progenitor cell masses proximally (lower double arrows in F)8. Scale bars 50 µm. Please click here to view a larger version of this figure.
Movie 1. Please click here to download this movie.
Movie 2. Please click here to download this movie.
Movie 3. Please click here to download this movie.
Movie 4. Please click here to download this movie.
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 separation of the epidermis from the dermis, which is technically challenging especially using hairy skin, and often results in tissue damage and rupture of the hair follicles. Also, separation of epidermis and dermis impairs studying the cellular and molecular interactions that occur between them during embryonic development and tissue homeostasis.
The 'flat mount approach' is another method where full thickness mouse skin is dissected and immunostained, and then clarified using benzyl benzoate and benzyl alcohol (BBBA) while preserving immunofluorescent signals14. This method does not require the technically challenging separation of the epidermis from the dermis. However, BBBA is highly toxic, it denatures some fluorescent proteins, and it contaminates microscope objectives, not allowing for the high-resolution imaging of the samples required to investigate cellular and molecular interactions in the tissue.
This report describes a CUBIC protocol5-7 to clarify full thickness mouse skin biopsies from any desired anatomical region and age using relatively safe and cheap reagents. This technically simple protocol allows visualization of protein expression and proliferation patterns by immunofluorescence in full thickness skin biopsies counterstained with DAPI in three dimensions at single cell resolution, enabling unprecedented and highly accurate qualitative assessment and quantification of protein expression, proliferation, tissue morphology and hair follicle dimensions. This method is also suitable for visualization of sebocytes using Nile Red staining despite the removal of tissue lipids during the clarification procedure. An important limitation of this method is the availability of antibodies efficiently binding and allowing visualization of their respective target proteins using this skin clarification process. In addition, the generated image and movies files are large, and good IT resources such as computers with powerful processors and ample data storage space are essential.
A critical step in the procedure is the preparation of the skin biopsies. To visualize intact hair follicle anatomy, the orientation of the skin biopsy should be taken into account, and the length of the biopsies should be cut parallel to the direction of the length of the hair follicles. In addition, small and very thin skin biopsies are difficult to mount correctly for microscopy. Larger biopsies will require more time to clear, and may incur antibody penetration problems resulting in antigen detection artefacts. It is therefore advisable to cut, clear and stain multiple biopsies per experiment.
It is also important to tightly seal the tubes while preparing the CUBIC solutions to prevent evaporation of the contents during the heating process, which results in changes in reagent concentrations and may cause impaired tissue clearing. The next important step is that the tissue will be clarified at 37 °C to accelerate the clearing process. After 7 days, the CUBIC1 solution will need to be prepared fresh to replace the old one.
In the future, more antibodies will be identified that are compatible with the CUBIC skin clarification procedure, and analyses may be extended to visualizing and quantification of epidermal and melanocyte (stem) cell markers and dermal proteins. This technique will also play a fundamental role in 3D studies of skin pathology and anatomy, and aid in elucidation of cellular and molecular mechanisms underlying abnormal and normal epidermal biology in skin of mutant mice and transgenic reporter mouse models, including signaling interactions between the dermis and the epidermis.
The CUBIC protocol5-7 is likely to be applicable to skin of other vertebrate animal models, and to human skin punch biopsies. Light sheet microscopy will enable visualization of epidermal anatomy, protein expression patterns, and progression of the hair follicle cycle stages in larger skin samples.
The authors have nothing to disclose.
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.
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 |