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 skin area with a trimmer taking care not to wound the skin.</li>
		<li>Wash skin to decontaminate with 70% ethanol in Phosphate Buffered Saline (PBS).</li>
		<li>Lift dorsal neck skin with forceps and make incision with scissors.</li>
		<li>Dissect a large area of dorsal mouse skin (approximately 1.5 x 4 cm).</li>
		<li>Flatten skin dermis-side down on a filter paper, and make note of the anterior-posterior orientation of the sample.</li>
		<li>Trim filter paper around the dissected skin, and place in a 15 ml tube filled with freshly prepared 4% paraformaldehyde (PFA)solution in PBS.</li>
		<li>Fix for 1 hr at room temperature, or overnight in the refrigerator at 4 &#176;C.</li>
		<li>Wash skin 2 x 5 min in PBS in a 15 ml tube. 
		Note: The following (1.1.10 - 1.1.12) are optional steps for long term storage of tissue.</li>
		<li>Dehydrate dissected skin in PBS with increasing concentration of ethanol (25%, 50% 70%)in a 15 ml tube during 1-hr wash steps at room temperature.</li>
		<li>Store dehydrated skin in 70% ethanol in PBS in a 15 ml tube at 4 &#176;C until further use.</li>
		<li>4 hours before clearing, rehydrate skin tissue in PBS with decreasing concentration of ethanol (70%, 50%, 25%, 0%) in a 15 ml tube during 1-hr wash steps at room temperature.</li>
	</ol>
	</li>
	<li>Clearing the mouse skin biopsies
	<ol>
		<li>Prepare the CUBIC1 clearing solution by dissolving 3.85 g urea and 3.85 g N,N,N&#39;,N&#39;-tetrakis (2-hydroxypropyl) ethylenediamine in 5.38 ml distilled water on a heater set to 60 - 70&#160;&#176;C. Use a hot stirrer.</li>
		<li>Add 2.31 g polyethylene glycol mono-p-isooctylphenyl ether/Triton X-100 to the solution once it is clear and has cooled down to room temperature.</li>
		<li>Cut the mouse skin with a sharp razor blade into biopsies of approximate dimensions 0.2 x 0.5 cm, and submerge in 5 ml of CUBIC1 clearing solution in a 15 ml tube. To optimize the visualization of hair follicles, ensure that the longer side of the biopsy is cut along the antero-posterior direction of the sample.</li>
		<li>Place on a rotating platform in a hybridization oven at 37 &#176;C.</li>
		<li>Change the clearing solution after 7 days. Prepare fresh CUBIC1 solution prior to use.</li>
		<li>Check the transparency of the tissue after 7 days. If necessary, leave biopsies in CUBIC1 clearing solution until the tissue is completely transparent.</li>
		<li>Once the skin biopsy is transparent, remove the CUBIC1 solution, and add 4 ml of 1&#215; PBS to wash the tissue 4 times for 6 hr&#160;at 37 &#176;C.</li>
		<li>Wash the skin tissue in 20% w/v sucrose in PBS in a 15 ml tube for 4 hr&#160;at 37 &#176;C.</li>
		<li>Freeze the tissue in mounting medium Optimal Cutting Temperature (OCT) Compound in a 15 ml tube overnight in a -80 &#176;C freezer. 
		Note: This step will increase the biopsy&#39;s permeability for antibody penetration in subsequent steps.</li>
	</ol>
	</li>
</ol>
2. Immunofluorescence Staining
<ol>
	<li>Thaw tissue from 1.2.9) to room temperature for 2 - 3 hr.</li>
	<li>Wash tissue in the 15 ml tube with 5 ml of PBS for 8 hr at room temperature to remove OCT Compound.</li>
	<li>Transfer biopsies to a 2 ml tube, and incubate tissue in 1 ml rabbit anti-Keratin14 or rabbit anti-Ki67 antibody, both diluted 1:100 in PBST (PBS + 0.1% Triton-X100) for 3 days on a shaker in a 37 &#176;C oven.</li>
	<li>Transfer biopsies to a 15 ml tube, and wash tissue 4 times for 6 hr in 5 ml of PBST on a shaker in a 37 &#176;C oven.</li>
	<li>Transfer biopsies to a 2 ml tube, add 1 ml anti-rabbit Alexa594 secondary antibody diluted 1:100 in PBST and incubate 3 days on a shaker in a 37 &#176;C oven.</li>
	<li>Transfer biopsies to a 15 ml tube, and wash tissue 4 times for 6 hr in 5ml of PBST on a shaker in a 37 &#176;C oven.</li>
	<li>Transfer biopsies to a 2 ml tube, add 1 ml 4&#39;,6-diamidino-2-phenylindole (DAPI) nuclear counterstain solution (1:1,000) in PBST and incubate overnight on a shaker in a 37 &#176;C oven.</li>
	<li>Remove DAPI counterstaining solution, and add 1 ml of PBST to the 2 ml tube to wash tissue 4 times for 6 hr on a shaker in a 37 &#176;C oven. 
	Note: Optional step: Biopsies can be stored in the dark in 1x PBS with 0.02% sodium azide for at least 3 weeks.</li>
</ol>
3. Nile Red Staining
<ol>
	<li>Dissolve Nile Red powder in dimethylsulfoxide (DMSO) to a final concentration of 1 mg/ml</li>
	<li>Add 1 &#181;l Nile Red solution to 1 ml PBS to a final concentration of 1 &#181;g/ml.</li>
	<li>Thaw tissue from 1.2.9) to room temperature for 2 - 3 hr, and wash with 5 ml PBS for 8 hr at room temperature.</li>
	<li>Transfer biopsies to a 2 ml tube, and submerge tissue in 1 ml Nile Red staining solution for 2.5 hr at room temperature.</li>
	<li>Wash the skin biopsies in the 2 ml tube with 1 ml PBST 4 times for 30 min at room temperature.</li>
	<li>Remove PBST solution from the 2 ml tube, add 1 ml of DAPI nuclear counterstaining solution (1:1,000) in PBST and incubate overnight at room temperature.</li>
	<li>Remove DAPI counterstaining solution, and add 1ml of PBST in the 2 ml tube to wash the skin tissue 4 times for 6 hr at room temperature. 
	Note: Optional step: Biopsies can be stored in the dark in 1x PBS with 0.02% sodium azide for at least 3 weeks.</li>
</ol>
4. Imaging
<ol>
	<li>Prepare the CUBIC2 clearing solution, which contains 50% (w/v) sucrose, 25% (w/v) urea, 10% (w/v) 2,2&#8242;,2&#8242;&#39;-nitrilotriethanol, and 0.1% (v/v) Triton X-100.</li>
	<li>Incubate the skin tissue in 1 ml CUBIC2 solution in a 2 ml tube on a shaker for 24 hr in a 37 &#176;C oven. This step will even the refractive index of the tissue.</li>
	<li>Check the clarity of the tissue. Once it is clear, position the entire skin biopsy (0.2 x 0.5 cm) on its longer side onto a glass coverslip, such that the direction of the length of the hair follicles is parallel to the coverslip surface (#1 24 x 60 mm) 
	Note: Optional step: Antibody-stained biopsies can be stored in CUBIC2 solution for about 7 days. Nile Red-stained biopsies can only be stored in CUBIC2 solution for up to 1 day, and should be imaged as soon as possible.</li>
	<li>Prepare imaging chamber (Figure 1) 
	Note: Consumables required for the preparation of the imaging chamber are: blue tack, play dough or similar, and 2 coverslips (24 x 50 mm) (Figure 1A).
	<ol>
		<li>Prepare two thin strips of blue tack (diameter approximately 1 mm x 2 cm), and two coverslips (Figure 1B).</li>
		<li>Place blue tack strips on one cover slip, allowing enough space for skin biopsy (Figure 1C). 4.4.3)</li>
		<li>Place skin biopsy in between strips on the coverslip in a drop of CUBIC2 solution (Figure 1C).</li>
		<li>Cover skin biopsy with the second coverslip (Figure 1D).</li>
	</ol>
	</li>
	<li>Place the imaging chamber with the mounted skin biopsy onto the stage of a confocal microscope and move the tissue into the light pathway.</li>
	<li>Scan the sample using a mercury or halogen light source and standard epifluorescence filters (e.g., DAPI/GFP/CY3/CY5) to identify fluorescently stained regions of interest.</li>
	<li>Image regions of interest using a 10X and 20X objective (NA 0.75) and standard confocal fluorescence imaging techniques (e.g., with DAPI stained samples illuminate with a 405 nm laser and collect fluorescence signal between 425 - 475 nm, and with Alexa Fluor 594 or Nile Red-stained samples illuminate with a 561 nm laser and collect fluorescence signal between 570 - 620 nm).</li>
	<li>Generate image Z-stacks of each region of interest using microscope Z-stack image acquisition software (e.g., using NIS elements imaging software 4.13 as described below).
	<ol>
		<li>Ensure optimal laser power and PMT HV/Offset settings have been selected to collect sample fluorescence.</li>
		<li>Open the &#34;ND Acquisition&#34; toolbar from &#34;Acquisition Controls&#34;.</li>
		<li>Select &#34;Z series Setup&#34; tab (ensure all other tabs are unselected).</li>
		<li>While live scanning, focus to the top of the sample and press the &#34;Top&#34; button.</li>
		<li>Focus to the bottom of the sample and press the &#34;Bottom&#34; button.</li>
		<li>Input required step size (or press optimized step size button).</li>
		<li>Press the &#34;Run now&#34; button.</li>
		<li>Save resultant Z-stacks as individual Tiff image stacks (1 fluorochrome per image Z-stack).</li>
	</ol>
	</li>
	<li>Use image Z-stacks to reconstruct 3D volume regions of interest using 3D analysis software (e.g., using Imaris x64 7.2.3 as described below).
	<ol>
		<li>Start analysis software.</li>
		<li>Choose &#34;Surpass&#34; button in toolbar for 3D volume generation.</li>
		<li>Open first color image Z-stack (e.g., DAPI).</li>
		<li>Use the &#39;add channel&#39; tool to add additional color channels, one channel per fluorochrome. Thus a sample containing both DAPI and Alexa Fluor 594 fluorochromes will require two channels.</li>
		<li>Use the &#34;Image Properties&#34; tool to set correct pixel (voxel) dimensions (XYZ), as determined in 4.7 above.</li>
		<li>Use &#34;Display Adjustment&#34; tool to change channel colors as needed (e.g., set DAPI channel to blue, Alexa Fluor 594 channel to red).</li>
		<li>To position the 3D volume in the image window, use the computer mouse to click and drag volume as required.</li>
		<li>Use the &#34;Snapshot&#34; tool in the toolbar to generate screen shots of the 3D image.</li>
		<li>Use the &#34;Animation&#34; tool in the toolbar to generate movies of 3D sample rotation.</li>
	</ol>
	</li>
</ol>

<ol><li>Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scratching+the+surface+of+skin+development%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Scratching the surface of skin development.</a> Nature. 445 (7130), 834-842 (2007).</li>
<li>Watt, F. M., Lo Celso, C., Silva-Vargas, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidermal+stem+cells%3A+an+update%5BTitle%5D)+AND+%22Curr+Opin+Genet+Dev%22%5BJournal%5D)">Epidermal stem cells: an update.</a> Curr Opin Genet Dev. 16 (5), 518-524 (2006).</li>
<li>Hardy, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+secret+life+of+the+hair+follicle%5BTitle%5D)+AND+%22Trends+Genet%22%5BJournal%5D)">The secret life of the hair follicle.</a> Trends Genet. 8 (2), 55-61 (1992).</li>
<li>Lim, X., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wnt+signaling+in+skin+development%2C+homeostasis%2C+and+disease%5BTitle%5D)+AND+%22Cold+Spring+Harb+Perspect+Biol%22%5BJournal%5D)">Wnt signaling in skin development, homeostasis, and disease.</a> Cold Spring Harb Perspect Biol. 5 (2), (2013).</li>
<li>Susaki, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.</a> Cell. 157 (3), 726-739 (2014).</li>
<li>Susaki, E. A., Tainaka, K., Perrin, D., Yukinaga, H., Kuno, A., Ueda, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+CUBIC+protocols+for+whole-brain+and+whole-body+clearing+and+imaging%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.</a> Nat Protoc. 10 (11), 1709-1727 (2015).</li>
<li>Tainaka, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-body+imaging+with+single-cell+resolution+by+tissue+decolorization%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Whole-body imaging with single-cell resolution by tissue decolorization.</a> Cell. 159 (4), 911-924 (2014).</li>
<li>Beverdam, A., Claxton, C., Zhang, X., James, G., Harvey, K. F., Key, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Yap+controls+stem%2Fprogenitor+cell+proliferation+in+the+mouse+postnatal+epidermis%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">Yap controls stem/progenitor cell proliferation in the mouse postnatal epidermis.</a> J Invest Dermatol. 133 (6), 1497-1505 (2013).</li>
<li>Fuchs, E., Green, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+keratin+gene+expression+during+terminal+differentiation+of+the+keratinocyte%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Changes in keratin gene expression during terminal differentiation of the keratinocyte.</a> Cell. 19 (4), 1033-1042 (1980).</li>
<li>Braun, K. M., Niemann, C., Jensen, U. B., Sundberg, J. P., Silva-Vargas, V., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Manipulation+of+stem+cell+proliferation+and+lineage+commitment%3A+visualisation+of+label-retaining+cells+in+wholemounts+of+mouse+epidermis%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis.</a> Development. 130 (21), 5241-5255 (2003).</li>
<li>Hamilton, E., Potten, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+hair+plucking+on+the+turnover+time+of+the+epidermal+basal+layer%5BTitle%5D)+AND+%22Cell+Tissue+Kinet%22%5BJournal%5D)">Influence of hair plucking on the turnover time of the epidermal basal layer.</a> Cell Tissue Kinet. 5 (6), 505-517 (1972).</li>
<li>Morris, R. J., Fischer, S. M., Slaga, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+that+a+slowly+cycling+subpopulation+of+adult+murine+epidermal+cells+retains+carcinogen%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Evidence that a slowly cycling subpopulation of adult murine epidermal cells retains carcinogen.</a> Cancer Res. 46 (6), 3061-3066 (1986).</li>
<li>Schweizer, J., Marks, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+developmental+study+of+the+distribution+and+frequency+of+Langerhans+cells+in+relation+to+formation+of+patterning+in+mouse+tail+epidermis%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">A developmental study of the distribution and frequency of Langerhans cells in relation to formation of patterning in mouse tail epidermis.</a> J Invest Dermatol. 69 (2), 198-204 (1977).</li>
<li>Chang, H., Wang, Y., Wu, H., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flat+mount+imaging+of+mouse+skin+and+its+application+to+the+analysis+of+hair+follicle+patterning+and+sensory+axon+morphology%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Flat mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology.</a> J Vis Exp. (88), e51749(2014).</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P6418</td><td></td></tr><tr><td>Ethanol 96% (undenaturated)</td><td>Chem-supply</td><td>UN1170</td><td></td></tr><tr><td>Nile Red</td><td>Sigma-Aldrich&amp;nbsp;</td><td>72485-100MG</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)</td><td>Roche</td><td>10236276001</td><td></td></tr><tr><td>&lt;em&gt;N,N,N&amp;rsquo;,N&amp;rsquo;&lt;/em&gt;-tetrakis (2-hydroxypropyl) ethylenediamine</td><td>Merck Millipore</td><td>821940</td><td></td></tr><tr><td>Polyethylene glycol mono-p-isooctylphenyl ether</td><td>Merck Millipore</td><td>648462</td><td></td></tr><tr><td>Triton X-100</td><td>Merck Millipore</td><td>648462</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S0389</td><td></td></tr><tr><td>Optimal Cutting Temperature (OCT) Compound</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>anti-Keratin14 antibody</td><td>Covance</td><td>PRB-155P</td><td></td></tr><tr><td>anti-Ki67 antibody&amp;nbsp;</td><td>Abcam</td><td>ab16667</td><td></td></tr><tr><td>Donkey anti-rabbit Alexa 594</td><td>Life Technologies</td><td>A21207</td><td></td></tr><tr><td>Dimethylsulfoxide</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D2650</td><td></td></tr><tr><td>Urea</td><td>Merck Millipore</td><td>66612</td><td></td></tr><tr><td>2,2&amp;prime;,2&amp;prime;&amp;rsquo;-nitrilotriethanol</td><td>Merck Millipore</td><td>137002</td><td></td></tr><tr><td>Confocal Microscope</td><td>Nikon Instruments Inc</td><td>Nikon A1 - Confocal Microscope</td><td></td></tr><tr><td>cruZer6 Face Trimmer</td><td>Braun</td><td>Braun cruZer6 Face</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich&amp;nbsp;</td><td>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 ...

cubic-protocol-visualizes-protein-expression-at-single-cell

Research

JoVE Journal

Biology

11.5K Views.  University of New South Wales Australia.  Dit rapport beschrijft een KUBIEKE protocol om de volledige dikte muis huid biopten te verduidelijken en te visualiseren eiwit expressie patronen, delende cellen, en sebocyten bij de enkele resolutie cel in 3D. Deze methode maakt een nauwkeurige evaluatie van de huid anatomie en pathologie, en abnormale epidermale fenotypes in genetisch gemodificeerde muis lijnen. 

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

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Developmental Biology

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein and RNA multiplexing by leveraging photo-cleavable oligo-tagged antibodies and probes, respectively. Oligos are cleaved and quantified from specific regions across the tissue to elucidate the underlying biology. Here, the study demonstrates that automated custom antibody visualization protocols can be utilized to guide ROI selection in conjunction with spatial proteomics assays. This specific method did not show acceptable performance with spatial transcriptomics assays. The protocol describes the development of a 3-plex immunofluorescent (IF) assay for marker visualization on an automated platform, using tyramide signal amplification (TSA) to amplify the fluorescent signal from a given protein target and increase the antibody pool to choose from. The visualization protocol was automated using a thoroughly validated 3-plex assay to ensure quality and reproducibility. In addition, the exchange of DAPI for SYTO dyes was evaluated to allow imaging of TSA-based IF assays on the spatial profiling platform. Additionally, we tested the ability of selecting small ROIs using the spatial transcriptomics assay to allow the investigation of highly-specific areas of interest (e.g., areas enriched for a given cell type). ROIs of 50 &#181;m and 300 &#181;m diameter were collected, which corresponds to approximately 15 cells and 100 cells, respectively. Samples were made into libraries and sequenced to investigate the capability to detect signals from small ROIs and profile-specific regions of the tissue. We determined that spatial proteomics technologies highly benefit from automated, standardized protocols to guide ROI selection. While this automated visualization protocol was not compatible with spatial transcriptomics assays, we were able to test and confirm that specific cell populations can successfully be detected even in small ROIs with the standard manual visualization protocol.

Here, we describe a protocol for fine-tuning regions of interest (ROIs) for Spatial Omics technologies to better characterize the tumor microenvironment and identify specific cell populations. For proteomics assays, automated customized protocols can guide ROI selection, while transcriptomics assays can be fine-tuned utilizing ROIs as small as 50 &#181;m.

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...

Cancer Research

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Bioengineering

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix.

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

Immunology and Infection

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 &#181;m to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 &#181;m depth) by confocal microscopy.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 &#181;m to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and ...

CUBIC-based Protein Expression Visualization: A Procedure for Visualizing Protein Expression in Full Thickness Mouse Skin Biopsies

Source: Liang, H. et al. <a href="https://www.jove.com/v/54401"> CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations</a>. J. Vis. Exp (2016)
This video describes the CUBIC-based visualization technique, a tissue clearing-dependent imaging technique for visual assessment of protein expression in whole-mount skin biopsies at single-cell resolution in 3D. This technically simple protocol allows the visualization of skin proteins without preparing skin sections.

Source: Liang, H. et al.  CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations. J. Vis. Exp (2016)
This ...

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

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