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 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 ...

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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 ...

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Biology

16.4K Views.  The Hospital for Sick Children. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in de vetbiologie veld, zoals hoe intercellulaire interacties en vetweefsel fysiologie veranderen op verschillende metabole voorwaarden zoals vasten en obesitas. Het belangrijkste voordeel van deze techniek is dat het optimaal behoudt de oorspronkelijke vet 3D-structuur en vermijdt lange verwerking stappen die meestal nodig zijn voor conventionele immunohistochemie. Hoewel deze methode inzicht kan geven in de structuur van he...

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

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

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

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

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

Developmental Biology

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Medicine

A Guide to Examining Intramuscular Fat Formation and its Cellular Origin in Skeletal Muscle

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build and maintain the extracellular matrix that acts as a molecular myofiber scaffold. In addition, FAPs are indispensable for myofiber regeneration as they secrete a multitude of beneficial factors sensed by the muscle stem cells (MuSCs). In diseased states, however, FAPs are the cellular origin of intramuscular fat and fibrotic scar tissue. This fatty fibrosis is a hallmark of sarcopenia and neuromuscular diseases, such as Duchenne Muscular Dystrophy. One significant barrier in determining why and how FAPs differentiate into intramuscular fat is effective preservation and subsequent visualization of adipocytes, especially in frozen tissue sections. Conventional methods of skeletal muscle tissue processing, such as snap-freezing, do not properly preserve the morphology of individual adipocytes, thereby preventing accurate visualization and quantification. To overcome this hurdle, a rigorous protocol was developed that preserves adipocyte morphology in skeletal muscle sections allowing visualization, imaging, and quantification of intramuscular fat. The protocol also outlines how to process a portion of muscle tissue for RT-qPCR, enabling users to confirm observed changes in fat formation by viewing differences in the expression of adipogenic genes. Additionally, it can be adapted to visualize adipocytes by whole-mount immunofluorescence of muscle samples. Finally, this protocol outlines how to perform genetic lineage tracing of Pdgfr&#945;-expressing FAPs to study the adipogenic conversion of FAPs. This protocol consistently yields high-resolution and morphologically accurate immunofluorescent images of adipocytes, along with confirmation by RT-qPCR, allowing for robust, rigorous, and reproducible visualization and quantification of intramuscular fat. Together, the analysis pipeline described here is the first step to improving our understanding of how FAPs differentiate into intramuscular fat, and provides a framework to validate novel interventions to prevent fat formation.

The replacement of healthy muscle tissue with intramuscular fat is a prominent feature of human diseases and conditions. This protocol outlines how to visualize, image, and quantify intramuscular fat, allowing the rigorous study of the mechanisms underlying intramuscular fat formation.

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

Immunology and Infection

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Human SATMVECs: Isolation and Adipocyte Cross-Talk Studies

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Author Spotlight: Insights into Cardiometabolic Diseases with Subcutaneous Adipose Tissue Microvasculature Studies 

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. ...

Three-Dimensional Adipocyte Culture as a Model to Study Cachexia-Induced White Adipose Tissue Remodeling

Cancer cachexia (CC) presents itself as a syndrome with multiple manifestations, causing a marked multi-organ metabolic imbalance. Recently, cachectic wasting has been proposed to be stimulated by several inflammatory mediators, which may disrupt the integrative physiology of adipose tissues and other tissues such as the brain and muscle. In this scenario, the tumor can survive at the host&#39;s expense. In recent clinical research, the intensity of depletion of the different fat deposits has been negatively correlated with the patient&#39;s survival outcome. Studies have also shown that various metabolic disorders can alter white adipose tissue (WAT) remodeling, especially in the early stages of cachexia development. WAT dysfunction resulting from tissue remodeling is a contributor to overall cachexia, with the main modifications in WAT consisting of morpho-functional changes, increased adipocyte lipolysis, accumulation of immune cells, reduction of adipogenesis, changes in progenitor cell population, and the increase of &#34;niches&#34; containing beige/brite cells.
To study the various facets of cachexia-induced WAT remodeling, particularly the changes&#160;progenitor cells and beige remodeling, two-dimensional (2D) culture has been the first option for in vitro studies. However, this approach does not adequately summarize WAT complexity. Improved assays for the reconstruction of functional AT ex vivo&#160;help the comprehension of physiological interactions between the distinct cell populations. This protocol describes an efficient three-dimensional (3D) printing tissue culture system based on magnetic nanoparticles. The protocol is optimized for investigating WAT remodeling induced by cachexia induced factors (CIFs). The results show that a 3D culture is an appropriate tool for studying WAT modeling ex vivo and may be useful for functional screens to identify bioactive molecules for individual adipose cell populations applications and aid the discovery of WAT-based cell anticachectic therapy.

This protocol describes a three-dimensional (3D) magnetic printing culture system that permits dissection of white adipose tissue (WAT) remodeling induced by a conditioned medium from cancer cells. Using a 3D culture system of UCP1+ adipocytes that express a green fluorescent protein (GFP) allows the study of beige adipocytes contributing to adipose tissue remodeling.

Cancer cachexia (CC) presents itself as a syndrome with multiple manifestations, causing a marked multi-organ metabolic imbalance. Recently, cachectic ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

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

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