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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scratching+the+surface+of+skin+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+stem+cells:+an+update.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secret+life+of+the+hair+follicle.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+in+skin+development,+homeostasis,+and+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis.">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. 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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 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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JoVE Journal

Biology

11.4K Views.  University of New South Wales Australia. 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.

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

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...