Name: cubic protocol visualizes protein expression at single cell resolution in whole mount skin preparations
Uploaded: 2016-08-04T15:00:00
Description: Watch this Scientific Journal Video about CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations at JoVE.com

We thank Australian Bio-Resources (Garvan Institute, Australia), the Biological Resources Centre (UNSW Australia) and the Animal Care &#38; 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.

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
<ol>
	<li>Collection of mouse skin tissue.
	<ol>
		<li>Humanely euthanize the mice by cervical dislocation.</li>
		<li>Carefully remove hairs from the relevant sk...

<ol>
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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...

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-&#916;C 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.

<table border="0" cellpadding="0" cellspacing="0" width="1082">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Paraformaldehyde</td>
			<td style="width:195px;">Sigma-Aldrich&#160;</td>
			<td style="width:229px;">P6418</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ethanol 96% (undenaturated)</td>
			<td style="width:195px;">Chem-supply</td>
			<td style="width:229px;">UN1170</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Nile Red</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>72485-100MG</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8217;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Roche</td>
			<td>10236276001</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N,N&#8217;,N&#8217;-tetrakis (2-hydroxypropyl) ethylenediamine</td>
			<td style="width:195px;">Merck Millipore</td>
			<td style="width:229px;">821940</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene glycol mono-p-isooctylphenyl ether</td>
			<td style="width:195px;">Merck Millipore</td>
			<td style="width:229px;">648462</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Triton X-100</td>
			<td style="width:195px;">Merck Millipore</td>
			<td style="width:229px;">648462</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sucrose</td>
			<td style="width:195px;">Sigma-Aldrich&#160;</td>
			<td style="width:229px;">S0389</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optimal Cutting Temperature (OCT) Compound</td>
			<td style="width:195px;">Tissue-Tek</td>
			<td style="width:229px;">4583</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">anti-Keratin14 antibody</td>
			<td style="width:195px;">Covance</td>
			<td style="width:229px;">PRB-155P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">anti-Ki67 antibody&#160;</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:229px;">ab16667</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:423px;">Donkey anti-rabbit Alexa 594</td>
			<td style="width:195px;">Life Technologies</td>
			<td style="width:229px;">A21207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dimethylsulfoxide</td>
			<td style="width:195px;">Sigma-Aldrich&#160;</td>
			<td style="width:229px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Urea</td>
			<td style="width:195px;">Merck Millipore</td>
			<td style="width:229px;">66612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,2&#8242;,2&#8242;&#8217;-nitrilotriethanol</td>
			<td style="width:195px;">Merck Millipore</td>
			<td style="width:229px;">137002</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:423px;">Confocal Microscope</td>
			<td style="width:195px;">Nikon Instruments Inc</td>
			<td style="width:229px;">Nikon A1 - Confocal Microscope</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">cruZer6 Face Trimmer</td>
			<td style="width:195px;">Braun</td>
			<td style="width:229px;">Braun cruZer6 Face</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Sodium azide</td>
			<td style="width:195px;">Sigma-Aldrich&#160;</td>
			<td style="width:229px;">438456</td>
			<td></td>
		</tr>
	</tbody>
</table>

cubic protocol visualizes protein expression at single cell resolution in whole mount skin preparations

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.

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...

Watch this Scientific Journal Video about CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations at JoVE.com

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

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 ...

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