This work was funded by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada, Pilot and Feasibility Study Grant of Banting &#38; Best Diabetes Centre (BBDC), the SickKids Start-up Fund to H-K.S., Medical Research Center Program (2015R1A5A2009124) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning to J-R.K.

All experimental animal protocols were approved by the Animal Care Committee of The Center for Phenogenomics (TCP) conformed to the standards of the Canadian Council on Animal Care. Mice were maintained on 12-h light/dark cycles and provided with free access to water and food. 7 month old C57BL/6J male mice were used in the whole-mount staining experiment.
NOTE: Sections 1 to 2 are in chronological order, with section 3 being an optional step right after section 1. Section 4 can be performed to analyze adipocyte size and blood vessel density after the completion of section 2.
1. Materials Preparation and Tissue Isolation
<ol>
	<li>Prepare fresh 1% paraformaldehyde (PFA) diluted in 1x phosphate buffered saline (PBS) for tissue fixation. Prepare 0.3% nonionic surfactant diluted in 1x PBS (hereafter referred to as PBS-0.3T) for subsequent tissue washing steps. 
	NOTE: For each tissue, prepare approximately 1.5 mL of 1% PFA to ensure complete immersion. Fixation volume may be increased or decreased depending on the tissue size. 
	CAUTION: This step is hazardous, as PFA is corrosive and toxic. Wear personal protective equipment (e.g., nitrile gloves, lab coat, footwear, safety goggles) and handle in a fume hood.</li>
	<li>Euthanize animals according to an approved procedure (e.g., cervical dislocation and/or carbon dioxide asphyxiation). Without delay, dissect the desired adipose tissue depots (e.g., inguinal white adipose tissue or perigonadal white adipose tissue)18.</li>
	<li>With dissection scissors, cut the tissue on a 100 x 15 mm2 Petri dish into pieces approximately 0.5&#8211;1 cm in size, and immerse them in microcentrifuge tubes filled with 1% PFA. Keep on ice.</li>
</ol>
2. Whole-mount Staining of White Adipose Tissue
<ol>
	<li>After dissection is complete, move the tissue samples in 1% PFA from the ice to room temperature (RT) for 1 h, and then transfer the tissues to a 12- or 24-well cell culture plate for quicker washing.</li>
	<li>Wash the tissues with PBS-0.3T, 3 times for 5 min each in RT on a shaker tilted at 22&#176;, with 20&#8211;25 tilts per min as the speed. 
	NOTE: Use this tilt and speed for all subsequent steps involving the use of a shaker. 
	NOTE: If using a multicolor reporter mouse line such as mT/mG and additional antibody staining is not needed, the tissue is ready for microscopy after step 2.2.</li>
	<li>Add 0.5&#8211;1 mL blocking buffer (5% animal serum diluted in PBS-0.3T). Put the plate on the shaker and incubate for 1 h in RT.</li>
	<li>Aspirate the blocking solution and add primary antibodies diluted in PBS-0.3T with 1% animal serum.</li>
	<li>Place the plate on a shaker at 4 &#176;C overnight.</li>
	<li>The next day, wash the tissues with PBS-0.3T 3 times for 5 minutes each in RT.</li>
	<li>Use appropriate secondary antibodies diluted in PBS-0.3T. Add 0.5&#8211;1 mL secondary antibody solutions to each well. Wrap the plate in aluminum foil and incubate it on a shaker for 1 hour at RT. 
	NOTE: Dilute the secondary antibody in the dark to prevent photobleaching.</li>
	<li>After secondary antibody incubation, wash with PBS-0.3T twice for 5 min, each in RT. Image the samples if a neutral lipid stain is not desired. If visualization of lipid droplets is needed using the neutral lipid stain, wash with 1x PBS (without non-ionic surfactant) twice, for 5 min each.</li>
	<li>After the washing steps, incubate with the neutral lipid stain diluted with 1:1500 dilution factor in 1x PBS for 30 min in RT. The tissues are now ready for microscopy. For future imaging, the tissues can be stored in this solution in 4 &#176;C. 
	NOTE: Imaging quality decreases over time; hence, the best time for imaging is within 1 or 2 days.</li>
	<li>Using forceps, lay the tissue flat on a 24 x 60 mm&#178; glass coverslip and place it on an inverted confocal laser microscope system.</li>
	<li>If staining of the nuclei with DAPI is desired, add 1&#8211;2 drops of mounting medium containing DAPI to completely submerge the tissue and prevent it from drying.</li>
	<li>To obtain images of whole-mount stained tissues at multiple focal planes, perform Z-stacks of 100&#8211;150 &#956;m in depth with 4&#8211;6 &#956;m step-size at the desired magnification.</li>
</ol>
3. Tissue Clearing and Immunolabeling Using iDISCO+
NOTE: This protocol is based on previously published procedures9,10,19.
<ol>
	<li>Fixation and methanol pre-treatment
	<ol>
		<li>Incubate the tissues in 4% PFA diluted in 1x PBS at 4&#176;C overnight in 2 mL microcentrifuge tubes. 
		NOTE: Leave the tissues in the 2 mL tubes for all the following treatments until imaging.</li>
		<li>The next day, wash the tissues in 1x PBS three times, for 1 hour each on a shaker at RT. 
		NOTE: This step can be a pausing point to leave the sample overnight at RT or 4 &#176;C.</li>
		<li>Dehydrate the tissues at RT in 20%, 40%, 60%, and 80% methanol, subsequently, for 1 h each. Dehydrate in 100% methanol at RT for 1 hour, then transfer the tissues to fresh 100% methanol and incubate at 4 C for 1 h. 
		NOTE: Dilute methanol in distilled water. During methanol incubation, there is no need to put samples on a shaker as long as the tissue samples are immersed. 
		CAUTION: This step is hazardous, as methanol is toxic. It is highly flammable upon open flames. Wear personal protective equipment (e.g., nitrile gloves, lab coat, safety goggles) and handle in a fume hood. Store the methanol away from ignition and in a flammable safety cabinet.</li>
		<li>Bleach the tissues with 5% hydrogen peroxide (H2O2; 1 volume of 30% H2O2 diluted in 5 volumes of 100% methanol) overnight at 4 &#176;C. 
		CAUTION: 30% hydrogen peroxide is very hazardous upon skin and eye contact. Wear personal protective equipment (e.g., nitrile gloves, lab coat, safety goggles) and handle in a fume hood.</li>
		<li>Rehydrate the tissues at RT in 80%, 60%, 40%, and 20% methanol and 1x PBS, subsequently, for 1 hour each.</li>
		<li>Wash with 0.2% nonionic surfactant diluted in 1x PBS twice, for 1 h each on a shaker at RT.</li>
	</ol>
	</li>
	<li>Immunolabeling 
	NOTE: Fill up the 2 mL tubes containing the tissue to the top of the tube with the solution used in each step to prevent tissue oxidation as soon as immunolabeling begins, until clearing is completed.
	<ol>
		<li>Permeabilize the tissues by incubating them in a solution of 1x PBS, 0.2% nonionic surfactant, 20% dimethyl sulfoxide (DMSO), and 0.3 M glycine at 37 &#176;C on an incubated tabletop orbital shaker for 2 days. 
		NOTE: The maximum incubation time for 37 &#176;C permeabilization step is 2 days.</li>
		<li>Block the tissues in a solution of 1x PBS, 0.2% nonionic surfactant, 10% DMSO, 5% donkey serum, and 1% Fc block at 37 &#176;C on an incubated tabletop orbital shaker for 2 days. 
		NOTE: The maximum incubation time for the blocking step is 2 days.</li>
		<li>Incubate the tissues in primary antibodies of interest in a solution of 1x PBS, 0.2% polysorbate 20, 10 &#181;g/mL heparin, 5% DMSO, and 5% donkey serum at 37 &#176;C on an incubated tabletop orbital shaker for 4 days.</li>
		<li>Wash with 1x PBS, 0.2% polysorbate 20, and 10 &#181;g/mL heparin on a shaker at RT five times, each for 1 h. 
		NOTE: This step can be a pause point to leave samples overnight in RT.</li>
		<li>Incubate tissues with secondary antibody in a solution of 1x PBS, 0.2% polysorbate 20, 10 &#181;g/mL heparin, and 5% donkey serum at 37 &#176;C on a tabletop orbital shaker for 4 days. 
		NOTE: From step 3.2.5, all samples need to be wrapped with aluminum foil to prevent photobleaching of secondary antibody.</li>
		<li>Wash tissues in a solution of 1x PBS, 0.2% polysorbate 20, and 10 &#181;g/mL heparin on a shaker in RT five times, for 2 h each. 
		NOTE: This step can be a pausing point to leave the samples overnight at RT.</li>
	</ol>
	</li>
	<li>Tissue clearing of white adipose tissue and volume imaging
	<ol>
		<li>Dehydrate the tissues contained in 2 mL tubes by incubating in 20%, 40%, 60%, and 80% methanol, subsequently, each for 1 hour at RT. Then, dehydrate the samples in 100% methanol twice at RT. 
		NOTE: This step can be a pausing point to leave your samples in 100% methanol overnight at RT.</li>
		<li>Incubate the tissues with a mixture of 2 volumes of DCM to 1 volume of methanol for 3 h at RT on a shaker. 
		NOTE: DCM is volatile. Make sure the tubes are tightly sealed to prevent evaporation. 
		CAUTION: This step is hazardous. DCM is toxic upon inhalation. Prolonged exposure can potentially cause chemical burns. Wear personal protective equipment (e.g., nitrile gloves, lab coat, footwear, safety goggles). Use a fume hood.</li>
		<li>Incubate the tissues in 100% DCM twice, for 15 min each on a shaker at RT.</li>
		<li>Incubate in 100% DBE in RT until imaging and for sample storage. Before imaging, invert the tubes several times to mix the solutions. 
		NOTE: Completely fill tubes with DBE to prevent oxidation, which can result in unsuccessful tissue clearing. Do not shake the tubes during DBE incubation. 
		CAUTION: This step is hazardous. DBE is toxic. It can cause irritation to the eyes and skin. Wear personal protective equipment (e.g., nitrile gloves, lab coat, safety goggles) and handle in a fume hood.</li>
		<li>Image the whole tissue sample with a light microscope that matches the refractive index of organic solvent DBE. Perform Z-stacking at a desired magnification and step-size for the whole tissue.</li>
	</ol>
	</li>
</ol>
4. Examples of Data Analysis from Whole-mount Stained Tissue Images Using ImageJ
NOTE: See https://imageJ.nih.gov/ij/download.html for download and installation instructions.
<ol>
	<li>Quantification of blood vessel density (Supplementary Figure 2)
	<ol>
		<li>Import the saved images of only the channel with blood vessel immunostaining onto ImageJ. 
		NOTE: The images for quantification should be consistent in terms of staining procedure and image acquisition condition. Identical reagents should be used. The exposure time, intensity, and magnification should also be equivalent in the imaging process20.</li>
		<li>Convert the image color into black-and-white for blood vessel density quantification. To do so, under &#8220;Image&#8221; tab, select the &#8220;Adjust&#8221; command, then the &#8220;Color Threshold&#8221; option. In the &#8220;Threshold Color&#8221; display box, choose &#8220;Dark Background&#8221;20 and select &#8220;B&#38;W&#8221; under &#8220;Threshold Color&#8221;.</li>
		<li>To measure the percentage of area of blood vessel signal against background, select the &#8220;Analyze&#8221; tab, then the &#8220;Analyze Particles&#8221; command. Under the display of &#8220;Analyzed Particles&#8221;, select the &#8220;Clear Results&#8221; and &#8220;Summarize&#8221; options. Click &#8220;OK&#8221;.</li>
		<li>Copy and paste the value of the percentage area under the summary tab into a spreadsheet for analysis. The percentage of area will indicate the blood vessel density. Repeat steps 4.3.1&#8211;4.3.4 for each individual image.</li>
	</ol>
	</li>
	<li>Quantification of adipocyte size21 (Supplementary Figure 3)
	<ol>
		<li>Import the saved images of the adipocytes onto ImageJ.</li>
		<li>To set the scale of the image, measure the length of the scale in pixels by tracing a line to the scale with a known distance on the image using the straight-line selection tool. Under the &#8220;Analyze&#8221; tab, select the &#8220;Set Scale&#8221; command. The distance of the line that was traced before will be automatically calculated in pixels.</li>
		<li>The &#8220;Set Scale&#8221; display box will appear. Enter the known distance and unit of length. Select &#8220;Global&#8221; to apply the scale setting for all imported images and click &#8220;OK&#8221;.</li>
		<li>To choose area as the method of measurement, under the &#8220;Analyze&#8221; tab select the &#8220;Set Measurements&#8221; command. A list of different options for measurements will appear. Select the &#8220;Area&#8221; option and click &#8220;OK&#8221;.</li>
		<li>Using the freehand selection tool, trace the perimeter of each adipocyte of interest. Under the &#8220;Analyze&#8221; tab, select the &#8220;Measure (Ctrl + M)&#8221; command, and the area of the adipocyte will appear. Repeat this procedure for other adipocytes in the image. 
		NOTE: To ensure accurate measurements, use multiple images for quantification.</li>
		<li>Copy and paste the area measurements into a spreadsheet for further data analysis.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Although conventional techniques such as histology and cryosection offer benefits for observing intracellular structure, whole-mount staining provides a different perspective in adipose tissue research, which enables 3D visualization of cellular architecture of minimally processed tissue.
In order to successfully perform whole-mount staining, the following suggestions should be taken into consideration. Different adipose tissue depots can yield various immunostaining results; thus, the type of adipose tissue depot used should be determined first. For instance, brown adipose tissue (BAT) is denser relative to white adipose tissue (WAT) due to smaller, multilocular adipocytes21. This increased density of BAT makes it difficult for antibodies to permeate through the tissue. In addition, the size of the adipose tissue is also imperative for proper antibody staining, since too large/thick tissues can also result in insufficient antibody penetration. Hence, when obtaining the adipose tissue during animal dissection, the distal portion of the perigonadal fat tissue should be used, since this is the thinnest region and can allow for sufficient antibody penetration and consistent data. Alternatively, for denser tissues, use of a fluorescent reporter mouse line, such as mT/mG, may allow for better visualization of the marker of interest, since this avoids the issue of insufficient antibody penetration. Subsequently, 1% PFA is used for tissue fixation to preserve the natural distribution of proteins and ensure tissue permeabilization and antibody penetration22,23. However, over-fixation can decrease antigen recognition and produce autofluorescence24. This is due to the reaction between aldehyde groups on PFA and other aldehyde-containing fixative and tissue components, which creates fluorescent compounds24. To prevent the need for an antigen-retrieval step, it is recommended to leave tissues in a fixative for only an hour at room temperature and begin the next steps as soon as fixation is completed7. Like many other immunolabeling techniques, titrating the antibody concentration is an essential troubleshooting step to ensure the desired signal.
Indeed, there are several limitations of whole-mount staining despite the aforementioned significance and advantages. Whole-mount staining has stringent requirements regarding tissue type and size, because insufficient antibody penetration and uneven staining can occur in thicker tissues such as muscle and liver. Also, whole-mount staining is not the most accurate representation of certain antibodies such as tyrosine hydroxylase (TH), a marker for the sympathetic nervous system, as its signal is often masked by dense lipid content in adipose tissue (Figure 7). In addition, the uneven surface of adipose tissue also posed a challenge for confocal imaging, since signals located at different layers of the adipose tissue cannot be easily captured on a single image. Therefore, the quantification of signal intensity in images obtained from whole-mount staining yield inconsistent results for whole-nerve fiber arborization. The distal portion of PWAT is usually the least lipid-dense; hence, better images can be obtained from this area. However, whether this area is representative of the entire tissue for immunostaining in all types of antibodies will require further investigation.
By making tissue optically transparent, a clearing method reduces light scattering, enabling visualization of the deep structure25. iDISCO+ is an inexpensive protocol recently developed that combines whole-mount immunolabeling with volume imaging of various large cleared tissues9. Modified iDISCO+ methods with LSFM demonstrated by recent studies9,10 observed dense dendritic arborization on WAT that cannot be seen with conventional immunolabeling and confocal microscopy. Conventional confocal imaging uses an imaging beam system and pinhole for laser scanning. The scanning speed and penetrating depth are limitations of the microscope; thus, 3D tissue dynamics are often missed. In contrast, light-sheet microscopy has apparent advantages in that it uses sheet-scanning and only illuminates one optical section at a time, capturing all the fluorescence molecules within that section. Moreover, fluorophores in other sections are not excited, preventing photo-bleaching and phototoxic effects26. Thus, the amount of nerve innervation within the adipose tissue can be more accurately assessed with iDISCO+ and LSFM. Despite this, there are some limitations to the use of iDISCO+ and LSFM. For instance, users should note that only channels in the red and far-red channel are compatible with iDISCO+, because longer wavelengths of light are better able penetrate the sample27. Additionally, autofluorescence in the blue-green spectrum is quite high in large tissue samples, so imaging in the red and far-red spectra will help reduce any autofluorescence that occurs14. In regard to transgenic fluorescent reporter mice, iDISCO+ can be used to visualize reporter proteins. However, immunolabeling of the fluorescent reporter with a secondary antibody should be conducted, as the endogenous signal may fade during the tissue clearing process14. Nonetheless, this technique is extremely valuable for studying sympathetic nervous system-adipose tissue interactions and for investigating adipose plasticity under different physiological and metabolic conditions.

Adipose tissue is an essential organ for energy storage and is characterized by dynamic remodelling and nearly unlimited expansion1. In addition to energy homeostasis, adipose tissue also plays an essential role in hormone secretion of over 50 adipokines to modulate whole-body metabolic function2. Adipose tissue has a diverse architecture comprising of various cell types including mature adipocytes, fibroblasts, endothelial cells, immune cells, and adipocyte progenitor cells3. Recent studies have shown that obesity and other metabolic dysfunction can significantly alter adipose tissue function and its microenvironment, which includes but is not limited to enlargement of adipocytes, infiltration of inflammatory cells (e.g., macrophages), and vascular dysfunction3.
Conventional morphological techniques such as histology and cryosectioning demonstrate several limitations in studying adipose biology such as lengthy chemical processing steps, which can lead to tissue shrinkage and structure distortion3,4. Furthermore, these 2D techniques are insufficient to observe intercellular interactions exerted by different cell types, as the sections obtained are limited to smaller regions of the entire tissue3. Compared to conventional methods of fluorescent imaging, whole-mount staining does not require additional invasive steps, such as embedding, sectioning, and dehydration; thus, this avoids the problem of diminishing antibody specificity. As such, it is a simple and efficient method for imaging adipose tissue, with better preservation of adipocyte morphology&#160;and overall adipose tissue structure5. Therefore, whole-mount staining as a quick and inexpensive immunolabeling technique was established to preserve adipose tissue 3D architecture1,6,7,8.
However, despite the preservation of adipose tissue morphology with use of whole-mount staining, this technique is still unable to visualize inner structures beneath the lipid surface of the tissue. Several recent studies9,10&#160;have established tissue clearing techniques combined with whole-mount immunolabeling1,6&#160;to allow for comprehensive 3D visualization within adipose tissue. In particular, dense neural and vasculature networks were visualized in recent studies9,10,11,12&#160;with 3D volume imaging. Indeed, studying the neural and vascular plasticity of adipose tissue under different physiological conditions is essential to study its biology. Immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO+) tissue clearing is a process comprised of methanol pre-treatment, immunolabeling, and clearing of tissue opaqueness with organic chemical reagents dichloromethane (DCM) and dibenzyl ether (DBE)13,14. By making the adipose tissue entirely transparent, a more accurate representation of anatomy within the tissue such as blood vessels and neural fibers can be obtained9,10. IDISCO+ has advantages in that it is compatible with various antibodies and fluorescent reporters11,14, and it has demonstrated success in multiple organs and even embryoes14. However, its main limitation is a long incubation time, in which 18 to 20 days are needed to complete the entire experiment.
Another important application of whole-mount staining is the visualization of cell fate in combination with a lineage tracing system. Lineage tracing is the labelling of a specific gene/marker in a cell that can be passed on to all daughter cells and is conserved over time15. As such, it is a powerful tool that can be used to determine the fate of a cell&#39;s progeny15. Since the 1990s, the Cre-LoxP recombinant system has become a powerful approach for lineage tracing in living organisms15. When a mouse line that expresses Cre, a DNA recombinase enzyme, is crossed with another mouse line expressing a reporter that is adjacent to a loxP-STOP-loxP sequence, the reporter protein is expressed15.
For whole-mount staining, the use of fluorescent multicolor reporters is suitable for imaging of adipose tissue because it allows for minimal interference with intracellular activities of the adipocyte16. However, traditional reporters typically stain the cytoplasm, making it difficult to trace the lineage of white adipocytes, which have limited cytoplasmic content17. To overcome this problem, the use of membrane-bound fluorescent tdTomato/membrane eGFP (mT/mG) reporter marker is an ideal tool. Membrane-targeted tdTomato is expressed in Cre-negative cells18. Upon Cre excision, a switch to the expression of membrane-targeted eGFP occurs, making this reporter suitable for tracing the lineage of adipocyte progenitors17,18&#160;(Supplementary Figure 1).
The purpose of this paper is to provide a detailed protocol for whole-mount staining and show how it can be combined with other techniques to study the development and physiology of adipose tissue. Two examples of applications described in this protocol are its use with 1) multicolor reporter mouse lines to identify various origins of adipocytes and 2) tissue clearing to further visualize the neural arborization in white adipose tissue (WAT).

<table>
	<tbody>
		<tr>
			<td>LipidTox</td>
			<td>Life Technologies</td>
			<td>H34477</td>
			<td></td>
		</tr>
		<tr>
			<td>PECAM-1 primary antibody</td>
			<td>Millipore</td>
			<td>MAB1398Z(CH)</td>
			<td></td>
		</tr>
		<tr>
			<td>TH (tyrosine hydroxylase) primary antibody</td>
			<td>Millipore</td>
			<td>AB152, AB1542</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI stain</td>
			<td>BD Pharmingen</td>
			<td>564907</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R confocal microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Confocal microscope</td>
		</tr>
		<tr>
			<td>Ultramicroscope I</td>
			<td>LaVision BioTec</td>
			<td></td>
			<td>Light sheet image fluorescent microscope</td>
		</tr>
		<tr>
			<td>Alexa Fluor secondary antibodies</td>
			<td>Jackson ImmunoResearch</td>
			<td></td>
			<td>Wavelengths 488, 594 and 647 used</td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse CD16/CD32</td>
			<td>BioSciences</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibenzyl-ether</td>
			<td>Sigma-Aldrich</td>
			<td>33630</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Chemical</td>
			<td>A452-1</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Hydrogen Peroxide</td>
			<td>BIO BASIC CANADA INC</td>
			<td>HC4060</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>J7126</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr>
			<td>Lectin kit I, fluorescein labeled</td>
			<td>VECTOR LABORATORIES</td>
			<td>FLK-2100</td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80</td>
			<td>Bio-Rad</td>
			<td>MCA497GA</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Hard Set Mounting Medium with DAPI</td>
			<td>VECTOR LABORATORIES</td>
			<td>H-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tween</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Animal serum (goat, donkey)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of 3d white adipose tissue structure using whole-mount staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

Due to the fragility of adipose tissue, methods involving multiple processing steps and sectioning can lead to disfigurement of adipose tissue morphology3 (Figure 1A). However, whole-mount staining can preserve the morphology of adipocytes, ensuring accurate interpretation of results (Figure 1B).
Over-fixation of adipose tissue leads to fixative-induced autofluorescence. As shown in Figure 2A, green and red channels staining for tyrosine hydroxylase (TH) and PECAM-1 signals, respectively, overlap in identical regions of the tissue, indicating that autofluorescence may have occurred due to over-fixation in PFA for 3 days. In contrast, Figure 2B shows a representative image of whole-mount staining when proper fixation is performed, as the signal for TH staining occurs in different areas relative to PECAM-1 signal, demonstrating that this signal is not autofluorescence and is in fact a positive signal.
Whole-mount staining is an important visualization tool for Cre-loxP-based lineage tracing of adipocytes15, with mT/mG being the ideal reporter system18. Ng2, a marker for adipocyte progenitor cell population, is a plasma membrane proteoglycan. In this system, Ng2-Cre-positive cells express m-GFP, whereas m-Tomato is expressed in Ng2-Cre-negative cells (Figure 3).
Adipose tissue is an incredibly dynamic organ, capable of expanding and shrinking under different physiological conditions and demands20. Imaging whole-mount stained adipose tissue allows for quantification of imorphological changes under different experimental conditions. In particular, adipose tissue is highly vascularized, which is important in mediating metabolic homeostasis upon rapid changes in energy level1. For instance, C57BL/6J mice that undergo 24 h of fasting display significantly smaller adipocyte size (Figure 4), indicating lipolysis, and a trend in elevated blood vessel density compared to continuously fed mice (Figure 5). ImageJ software was utilized to quantify the size of the adipocytes and blood vessel density, as described above.
Tissue clearing is a relatively new technique developed to remove the opaqueness of adipose tissue to allow visualization deep within the tissue volume9,10 (Figure 6). Whole-mount staining on uncleared IWAT using confocal microscopy only showed sparse sympathetic innervation, since nerve fibers underneath the surface of the tissue could not be visualized (Figure 7A). However, dense neural arborization could be observed after tissue clearing and immunolabeling with the use of iDISCO+ as well as the use of light-sheet fluorescent microscopy (LSFM) (Figure 7B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58683/58683fig1.jpg" /> 
Figure 1: Comparison of adipose tissue morphology with conventional morphological techniques and whole-mount staining technique. (A) H&#38;E stained adipose tissue on a paraffin-embedded section (left), with black arrowheads indicating distorted regions of adipocytes. Lectin (carbohydrate binding protein, white arrows) fluorescent dye injection, immunofluorescent staining of F4/80 (macrophage marker, yellow arrowheads), and DAPI nuclei staining of adipose tissue on cryosection (right). (B) White adipose tissue visualization using whole-mount staining with a step-size of 5 &#956;m. The total Z-stack depth captured is around 100 &#956;m. Adipocyte lipid droplets were stained with neutral lipid stain (grey), and blood vessels were stained with PECAM-1. Image was captured with microscopy with a step-size of 5 &#956;m for Z-stacking (red). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58683/58683fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58683/58683fig2.jpg" /> 
Figure 2: Visualization of neural fibers and blood vessels using whole-mount stained adipose tissue. (A) Representative microscopic images of undesirable results from whole-mount stained PWAT due to over-fixation in PFA for 3 days. Overlapping signals are indicated by white arrowheads. (B) Representative microscopic images of a positive result from whole-mount stained IWAT from control mouse. The images were captured at 100X magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58683/58683fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58683/58683fig3.jpg" /> 
Figure 3: Lineage tracing using mT/mG system in the adipose tissue. Representative images Cre-positive (mG) and Cre-negative (mT) cells in IWAT of a Ng2-Cre; mT/mG mouse. The images were captured at 200X magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58683/58683fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58683/58683fig4.jpg" /> 
Figure 4: Visualization and quantification of adipocyte size. (A) Representative images of adipocytes in PWAT of fed and 24-hour fasted C57BL/6J mice using neutral lipid staining. The images were captured at 200x magnification. (B) Adipocyte size comparison between fed and 24-h fasted C57BL/6J mice using ImageJ software. Values are expressed as mean &#177; SEM; 2-tailed unpaired Student&#8217;s t-test; ***p &#60; 0.001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58683/58683fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58683/58683fig5.jpg" /> 
Figure 5: Visualization and quantification of blood vessel density. (A) Representative images of blood vessels in fed and 24-hour fasted C57BL/6J mice using PECAM-1 antibody. (B) Comparison of blood vessel density between fed and 24-hour fasted C57BL/6J mice using ImageJ software. Values are expressed as mean &#177; SEM; 2-tailed unpaired Student&#8217;s t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58683/58683fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58683/58683fig6.jpg" /> 
Figure 6: Clearing of adipose tissue using the iDISCO+ method. Prior to clearing, the tissue is opaque. The tissue becomes completely transparent at the end of the tissue clearing steps.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58683/58683fig7.jpg" /> 
Figure 7: Whole-mount stained IWAT compared with tissue-cleared IWAT using iDISCO+ method. (A) Visualization of neural fibers using TH antibody (1:500) in whole-mount stained IWAT at 100x magnification with confocal microscopy with a step-size of 5 &#956;m. The total Z-stack depth captured is around 100 &#956;m. (B) Visualization of neural fibers using TH antibody (1:200) in whole-mount stained IWAT with iDISCO+ protocol at 1.6X magnification using LSFM with a step-size of 4 &#956;m. The total Z-stack depth captured is around 8 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58683/58683fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 1" src="/files/ftp_upload/58683/58683sfig1.jpg" /> 
Supplementary Figure 1: Schematic diagram for mT/mG lineage tracing system. A dual fluorescent system that employs membrane-targeted eGFP and membrane-targeted tdTomato. Before Cre recombination, the mT is globally expressed. When the cell expresses Cre, the mT cassette is excised, and mG is expressed permanently. pA represents polyadenylation sequences after the stop codon. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58683/58683sfig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 2" src="/files/ftp_upload/58683/58683sfig2.jpg" /> 
Supplementary Figure 2: Application of ImageJ software to quantify percentage area of blood vessel density. (A) &#8220;Image&#8221; tab commands and options to convert an image into a black-and-white threshold color. (B) Summary measurement of percentage area of vessel density using the &#8220;Analyze Particles&#8221; command. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58683/58683sfig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 3" src="/files/ftp_upload/58683/58683sfig3.jpg" /> 
Supplementary Figure 3: Application of ImageJ software to quantify adipocyte area. &#8220;Analyze&#8221; tab: &#8220;Set Scale&#8221; and &#8220;Measurement&#8221; commands to measure the area of the adipocyte(s). Results display shows the area of each adipocyte measured. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58683/58683sfig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining at JoVE.com

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

visualization-3d-white-adipose-tissue-structure-using-whole-mount

Nanyang Technological University

Research

JoVE Journal

Biology

16.2K Views.  The Hospital for Sick Children. The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Video: Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

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  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

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

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.

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 ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

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 ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

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.

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 ...

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the biliary tract in Suncus murinus (S. murinus&#160;). First, the specimen was dissected from S. murinus and fixed in 4% paraformaldehyde (PFA). Second, an enzymatic treatment and potential endogenous peroxidase inactivation were performed. The specimen was then exposed to the primary antibody, anti-neurofilament protein antibody, for 3-6 days. It was then incubated with the secondary antibody conjugated with horseradish peroxidase. The color reaction was revealed by reacting the specimen with a 3,3&#39;-diaminobenzidine (DAB) substrate. This method can be applied to analyze the innervation of all visceral organs. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be done first. This method was originally introduced by Kuratani and Tanaka1,2,3.

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

A whole-mount immunohistochemical approach, to visualize neurofilament protein expression in the extrahepatic biliary tract in Suncus murinus. is presented here. This protocol can be used to analyze the innervation of all visceral organs in S. murinus or other species.

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

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

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus