This work was supported by INSERM, Universit&#233; C&#244;te d&#8217;Azur, and by grants from the French National Research Agency (ANR) through the Investments for the Future Labex SIGNALIFE (ANR-11-LABX-0028-01), the program UCA JEDI (ANR-15-IDEX-01) via Academy 2 &#8220;Syst&#232;mes Complexes&#8221; and Academy 4 &#8220;Complexit&#233; et diversit&#233; du vivant&#8221;, Fondation pour la Recherche M&#233;dical (&#201;quipe FRM DEQ20180839587), and the Young Investigator Program to J.G. (ANR18-CE14-0035-01-GILLERON). We also thank the Imaging Core Facility of C3M funded by the Conseil D&#233;partemental des Alpes-Maritimes and the R&#233;gion PACA, and which is also supported by the IBISA Microscopy and Imaging Platform C&#244;te d&#8217;Azur (MICA). We thank Marion Dussot for technical help in tissue preparation. We thank Abby Cuttriss, UCA International Scientific Visibility, for proof reading of the manuscript.

This protocol was tested and is validated for all mouse and human white adipose tissue depots. Human and mouse adipose tissues were collected accordingly to European laws and approved by French and Swedish Ethical committees.
1. Fixation of mouse and human white adipose tissue
<ol>
	<li>Immerse the harvested mouse or human white adipose tissues in at least 10 mL of PBS containing 4% paraformaldehyde (PFA) in a 15 mL plastic tube.</li>
	<li>Shake the plastic tube at room temperature on a rolling plate for 1 h.</li>
	<li>Leave the plastic tube at 4 &#176;C on a rolling plate overnight, to complete the fixation. 
	NOTE: This protocol is adapted for large adipose tissue samples, such as whole epididymal fat pads obtained from mice fed a normal diet (&#8776;250 mg - &#8776;0.6 cm3). For larger samples like epididymal fat pads obtained from mice fed a high fat diet (&#8776;1.5 g - &#8776;4 cm3) or human adipose tissue samples, although the clearing protocol should perfectly work for the whole sample (by scaling-up the PFA and the antibody mixtures), in general, we cut tissue pieces around 1 g (&#8776;2.5 cm3) to reserve the remaining samples either for additional staining or applications. For the PFA (step 1.1.) we recommend using roughly 10 times the volume of the tissue. For the antibody mixtures (steps 3.1. and 3.4.), increase the volume to completely immerse the tissue, and use a 15 mL plastic tube (see Table of Materials) if the tissue is too large for a 1.5 mL plastic microtube (see Table of Materials).</li>
</ol>
2. Permeabilization and saturation of mouse and human white adipose tissue
<ol>
	<li>Rinse the fixed white adipose tissue in 10 mL of PBS for 5 min at room temperature to remove all traces of PFA.</li>
	<li>Immerse the tissue in a 15 mL plastic tube containing 10 mL of PBS supplemented with 0.3% glycine (see Table of Materials) and shake the tube at room temperature in an orbital shaker for 1 h at 100 revolutions per minute (rpm) to quench the remaining free aldehyde groups.</li>
	<li>Immerse the tissue in a 15 mL plastic tube containing 10 mL of PBS supplemented with 0.2% Triton X-100 (see Table of Materials) and shake the tube at 37 &#176;C in a temperature-controlled orbital shaker for 2 h at 100 rpm.</li>
	<li>Immerse the tissue in a 15 mL plastic tube containing 10 mL of PBS supplemented with 0.2% Triton X-100 and 20% DMSO (see Table of Materials) and shake the tube at 37 &#176;C in a temperature-controlled orbital shaker at 100 rpm overnight.</li>
	<li>Immerse the tissue in a 15 mL plastic tube containing 10 mL of PBS supplemented with 0.1% Tween-20 (see Table of Materials), 0.1% Triton X-100, 0.1% deoxycholate (see Table of Materials) and 20% DMSO and shake the tube at 37 &#176;C in a temperature-controlled orbital shaker at 100 rpm for at least 24 h.</li>
	<li>Rinse the tissue in a 15 mL plastic tube containing 10 mL of PBS supplemented with 0.2% Triton X-100 and shake the tube at room temperature in an orbital shaker at 100 rpm for 1 h.</li>
	<li>Immerse the tissue in a 15 mL plastic tube containing 10 mL of PBS supplemented with 0.2% Triton X-100, 10% DMSO and 3% BSA (see Table of Materials) and shake the tube at 37 &#176;C in a temperature-controlled orbital shaker at 100 rpm for 12 h, to saturate any sites that could non-specifically bind antibodies. 
	NOTE: In step 2.7, the BSA can be substituted by blood serum of the species of the secondary antibody used.</li>
</ol>
3. Staining procedure for mouse and human white adipose tissue
<ol>
	<li>Transfer the tissue in a 1.5 mL plastic microtube containing 300 &#181;L of PBS supplemented with 0.2% Triton X-100, 10% DMSO, 3% BSA and the primary antibodies (10x more concentrated than for cryosection staining but optimal antibody concentration should be evaluated for each antibody). Protect the tube from light by covering with aluminium foil and shake the tube at 37 &#176;C in a temperature-controlled orbital shaker at 100 rpm for at least two&#160;days (see the note in step 1.1 and step 3.7.1).</li>
	<li>Rinse the tissue in a 15 mL plastic tube containing 10 mL of PBS supplemented with 0.2% Triton X-100, 10% DMSO and 3% BSA and shake the tube protected from light at 37 &#176;C in a temperature-controlled orbital shaker at 100 rpm for 5 h. Perform this step twice.</li>
	<li>Rinse the tissue in a 15 mL plastic tube containing 10 mL of PBS supplemented with 0.2% Triton X-100, 10% DMSO and 3% BSA and shake the tube, protected from light, at 37 &#176;C in a temperature-controlled orbital shaker at 100 rpm for one night to two days.</li>
	<li>Transfer the tissue to a 1.5 mL plastic microtube containing 300 &#181;L of PBS supplemented with 0.2% Triton X-100, 10% DMSO, 3% BSA and the secondary antibodies (10x more concentrated than for cryosection staining). Protect the tube from light by covering with aluminium foil and shake the tube at 37 &#176;C in a temperature-controlled orbital shaker at 100 rpm for at least two&#160;days (see the note in step 1.1 and step 3.7.1).</li>
	<li>Rinse the tissue in a 15 mL plastic tube containing 10 mL of PBS supplemented with 0.2% Triton X-100, 10% DMSO, and 3% BSA and shake the tube, protected from light, at 37 &#176;C in a temperature-controlled orbital shaker at 100 rpm for 5 h. Perform this step twice.</li>
	<li>Rinse the tissue in a 15 mL plastic tube containing 10 mL of PBS supplemented with 0.2% Triton X-100, 10% DMSO, and 3% BSA and shake the tube, protected from light, at 37 &#176;C in a temperature-controlled orbital shaker at 100 rpm for one night to two days.</li>
	<li>Rinse the tissue in a 15 mL plastic tube containing 10 mL of PBS and shake the tube, protected from light, at 37 &#176;C in a temperature-controlled orbital shaker at 100 rpm for 2 h. 
	NOTE: For the labelling of specific structures such as nuclei or actin, add 4&#8242;,6-diamidino-2-phenylindole (DAPI or equivalent) and/or the fluorescently labelled-phalloidin have to be added at step 3.1. when only fluorescently labelled primary antibodies are used, or at step 3.4. when secondary antibodies are used. 
	NOTE: For the staining procedure, primary antibodies already conjugated with fluorochromes (like those used for flow cytometry) can be used and we recommend it for the gain of time and specificity. Indeed, even if mouse primary antibodies can be used followed by secondary anti-mouse antibodies, as we demonstrated it here, we have often observed non-specific staining of blood vessels due to circulating immunoglobulin. Therefore, to avoid this issue, primary antibodies that are already labelled are highly recommended and here we provide evidence that the antibodies used in flow cytometry are compatible with our procedure. However, in the specific case of an antigen that is expressed at low levels, a signal amplification step via secondary antibodies is mandatory; when the only available primary antibody is made in mouse, a cardiac-perfusion of the mice with PBS for at least 5 min at the sacrifice can remove a large proportion of circulating immunoglobulin and thus the non-specific staining.</li>
</ol>
4. Clearing procedure for mouse and human white adipose tissue
<ol>
	<li>Immerse the tissue in a 15 mL plastic tube containing 10 mL of 50% ethanol and shake the tube, protected from light, at room temperature in an orbital shaker at 100 rpm for 2 h.</li>
	<li>Immerse the tissue in a 15 mL plastic tube containing 10 mL of 70% ethanol and shake the tube, protected from light, at room temperature in an orbital shaker at 100 rpm for 2 h.</li>
	<li>Immerse the tissue in a 15 mL plastic tube containing 10 mL of 95% ethanol and shake the tube, protected from light, at room temperature in an orbital shaker at 100 rpm for 2 h.</li>
	<li>Immerse the tissue in a 15 mL plastic tube containing 10 mL of 100% ethanol and shake the tube, protected from light, at room temperature in an orbital shaker at 100 rpm for 2 h.</li>
	<li>Immerse the tissue in a 15 mL plastic tube containing 10 mL of 100% ethanol and shake the tube, protected from light, at room temperature in an orbital shaker at 100 rpm overnight.</li>
	<li>Immerse the tissue in a 20 mL glass bottle with a plastic cap (see table of materials) containing 5 mL of methyl salicylate (see Table of Materials) under a chemical hood and shake the glass container, protected from light, at room temperature in an orbital shaker at 100 rpm for at least 2 h. 
	NOTE: The clearing procedure could be significantly accelerated (if necessary) by avoiding steps 4.3. and 4.5., although the final clearing quality would be slightly reduced.</li>
</ol>
5. 3D-confocal imaging of cleared white adipose tissue
<ol>
	<li>Transfer the tissue to a metallic imaging chamber equipped with a glass bottom (see Table of Materials) under a chemical hood and fill the chamber with fresh methyl salicylate. 
	NOTE:&#160;To secure the tissue in place, and thus to prevent it from floating or moving sideways in the chamber, apply multiple 18 mm round glass coverslips (see Table of Materials) on top of the tissue when mounting it into the chamber.</li>
	<li>Place the imaging chamber on an inverted confocal microscope.</li>
	<li>Image the tissue using a low magnification objective (e.g., 4x objective) to generate a few cm3 3D maps of the whole adipose tissue or of the human tissue sample.</li>
	<li>Select several areas for the tissue sampling at higher magnification. Typically, use a 20x long distance air objective that provides a good ratio between resolution and depth. We acquire large mosaic images with z-stacks between 600 and 2000 &#181;m depth. 
	NOTE:&#160;Use a long-distance objective to image deeper into the tissue.</li>
</ol>
6. Extraction of quantitative results from the 3D adipose tissue images
NOTE: The segmentation of the different structures and the subsequent extraction of the quantitative information from the 3D-image stack generated in point 5 can be performed using any of the many existing image analysis software options, either commercial or freeware. In the following points, we describe a strategy that is routinely used in our laboratory to extract quantitative information from 3D adipose tissue images using commercial software (see Table of Materials).
<ol>
	<li>Convert the 3D stacks onto the software format to free-up memory space.</li>
	<li>Segment the cells.
	<ol>
		<li>Open the Cell module of the software.</li>
		<li>Change the Cell Detection setting to plasma membrane staining.</li>
		<li>Choose the fluorescent channel of the marker used to delineate the cell periphery (phalloidin which labels cortical actin or plasma membrane markers such as F4/80 for macrophage, TCR-&#946; for T cells, or cluster of differentiation CD proteins specific for immune cell subtypes).</li>
		<li>Set up the thresholds and provide a range of expected cell size (i.e., 1 to 200 &#181;m for cell detection).</li>
		<li>Run the segmentation. A volume corresponding to the z-stack will be generated with the segmented cells color-coded with a different color for each of the neighbouring cells.</li>
		<li>Apply statistical filters (size, roundness, circularity, etc.) in the statistic tab to exclude segmentation artefacts and/or to refine the segmented cells to the cell of interest based on their size (i.e., 20-200 &#181;m for adipocytes; 5-25 &#181;m for macrophages; 1-3 &#181;m for lymphocytes).</li>
		<li>Extract measurements and quantitative data (volume, number, size, diameter, etc.) from the statistics tab.</li>
	</ol>
	</li>
	<li>Segment cellular components (nuclei, vesicles, etc.) or vessels as a structure. 
	NOTE: Although the filament segmentation is very useful to study the connectivity of the vessels, we use the surface module segmentation to extract data on the size, volume and diameters of the vessels. Indeed, the quantification of the sizes of the vessels is lost when using the filaments module.
	<ol>
		<li>Open the Surface module of the software.</li>
		<li>Select the fluorescent channel of the marker used to specifically label the subcellular component to reconstruct it in 3D.</li>
		<li>Set up the thresholds and provide a range of the expected diameter size of the structure (i.e., 0.5-5 &#181;m for nuclei; 0-100 &#181;m for vessels; etc.).</li>
		<li>Run the segmentation. A volume with the segmented cellular structure will be generated. Measurements and quantitative data (volume, number, size, diameter, etc.) can be extracted from the statistics tab.</li>
	</ol>
	</li>
	<li>Segment the vessels as a tubular continuum. 
	NOTE: Although the filament segmentation is very useful to study the connectivity of the vessels, we use the surface module segmentation to extract data on the size, volume and diameters of the vessels. Indeed, the quantification of the sizes of the vessels is lost when using the filaments module.
	<ol>
		<li>Open the Filaments module of the software.</li>
		<li>Select the fluorescent channel corresponding to the vessel staining.</li>
		<li>Set up the thresholds for the fluorescence intensity and select the appropriate number of expected connecting nodes.</li>
		<li>Run the segmentation. Filaments representing vessel network will be generated in 3D. Measurements can be extracted from the statistics tab, allowing to obtain quantitative data including vessel length, number of vessel branching, etc.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts to disclose.

The modifications that occur within the adipose tissue over the course of pathological progression, such as that of obesity, is fundamental to the understanding of the mechanisms behind the pathology. Pioneering studies that revealed such mechanisms in adipose tissue have been based on global approaches such as whole adipose tissue proteomics21, flow cytometry22,23, and transcriptomics24,<sup class="xref"...

Obesity is characterized by an increase in adipose tissue mass and has become a major worldwide public health issue, given that people with obesity have increased risk of developing cardiovascular disease, type-2 diabetes, liver diseases and some cancers.
A fundamental physiological function of adipose tissue is to modulate whole-body glucose and lipid homeostasis1,2. During the feeding period, the adipocytes (i.e., the main cells of the adipose tissue) store&#160;the excess of glucose and lipids provided by a meal into triglycerides. During fasting, the adipocytes break down the triglycerides into non-esterified fatty acids and glycerol to sustain the energy demand of the body. During the development of obesity, adipose tissue expand by increasing the size (hypertrophia) and/or the number (hyperplasia) of adipocytes1, to increase their storage capacity. When the expansion of adipose tissue reaches its limit, a constant highly variable among patients, the remaining lipids accumulates into other metabolic organs including muscles and liver3,4, leading to their functional failure and initiating obesity-related cardio-metabolic complications1,5. Therefore, identifying the mechanisms that govern adipose tissue expansion is a key clinical challenge.
The morphological modifications documented within adipose tissues during obesity are linked to its pathological dysfunction. Several staining procedures have been used to describe the tissue organization of the adipose tissue, including actin6, vascular markers7, lipid-droplet markers8, and specific immune cell markers9,10. However, because of the huge diameter of adipocytes (50 to 200 &#181;m)11, it is essential to analyze a large portion of the whole tissue in three dimensions in order to accurately analyze the dramatic structural AT changes observed during obesity. However, because the light does not penetrate an opaque tissue, imaging in 3D within a large tissue samples using fluorescence microscopy is not possible. Methods of tissue clearing to make them transparent have been reported in the literature (for a review, see12) allowing one to clear tissues and to perform in-depth, whole tissue fluorescence microscopy. These methods offer unprecedented opportunities to assess the 3D cellular organization in healthy and diseased tissue. Each of the described methods have advantages and drawbacks, and therefore need to be carefully selected depending on the studied tissue (for a review, see13). Indeed, some approaches require a long incubation period and/or the use of materials or compounds that are either expensive, toxic or difficult to obtain14,15,16,17,18,19. Taking advantage of one of the first compounds used a century ago by Werner Spalteholz to clear tissues20, we set up a user-friendly and inexpensive protocol that is very well adapted for the clearing of all mouse and human adipose tissue depots in any laboratory with typical equipment including a chemical hood, a temperature-controlled orbital shaker and a confocal microscope.

<table><tbody><tr><td>1.5 mL microtubes</td><td>Eppendorff tubes - Dutscher</td><td>33528</td><td></td></tr><tr><td>15 mL plastic tubes</td><td>Falcon tubes - Dutscher</td><td>352096</td><td></td></tr><tr><td>18 mm round glass coverslip</td><td>Mariendfeld</td><td>0117580</td><td></td></tr><tr><td>20 mL glass bottle</td><td>Wheaton</td><td>986546</td><td></td></tr><tr><td>anti-mouse-alexa647-conjugated antibody</td><td>Jackson ImmunoResearch</td><td>715-605-150</td><td>Dilution: 1/100</td></tr><tr><td>anti-rabbit-alexa647-conjugated antibody</td><td>Jackson ImmunoResearch</td><td>711-605-152</td><td>Dilution: 1/100</td></tr><tr><td>BSA</td><td>Sigma-aldrich</td><td>A6003</td><td></td></tr><tr><td>CD301-PE antibody</td><td>Biolegend</td><td>BLE145703</td><td>Dilution: 1/100</td></tr><tr><td>CD31 antibody</td><td>AbCam</td><td>ab215912</td><td>Dilution: 1/50</td></tr><tr><td>Commercial 3D analysis software - IMARIS</td><td>Oxford instrument</td><td></td><td>with Cell module</td></tr><tr><td>Confocal microscope - Nikon A1R</td><td>Nikon</td><td></td><td></td></tr><tr><td>Dapi</td><td>ThermoFisher</td><td>D1306</td><td>Stock Concentration: 5 mg/mL; dilution 1/1000</td></tr><tr><td>Deoxycholate</td><td>Sigma-aldrich</td><td>D6750</td><td></td></tr><tr><td>DMSO</td><td>Sigma-aldrich</td><td>D8418</td><td></td></tr><tr><td>Glut4 antibody</td><td>Santa Cruz</td><td>sc-53566</td><td>Dilution: 1/50</td></tr><tr><td>Glycine</td><td>Sigma-aldrich</td><td>G7126</td><td></td></tr><tr><td>Lectin-DyLight649</td><td>Vector Lab</td><td>DL-1178-1</td><td>Stock Concentration : 2 &amp;micro;g/&amp;micro;L; IV Injection: 50 &amp;micro;L/mice</td></tr><tr><td>Metallic imaging chamber equipped with glass bottom - AttoFluor Chamber</td><td>Thermofisher</td><td>A7816</td><td></td></tr><tr><td>Methyl salicylate</td><td>Sigma-aldrich</td><td>M6752</td><td></td></tr><tr><td>Perilipin antibody</td><td>Progen</td><td>651156</td><td>Dilution: 1/50</td></tr><tr><td>Phalloidin-alexa488</td><td>ThermoFisher</td><td>A12379</td><td>Dilution: 1/100</td></tr><tr><td>TCR-&amp;beta;-PB antibody</td><td>Biolegend</td><td>BLE109225</td><td>Dilution: 1/100</td></tr><tr><td>TH antibody</td><td>AbCam</td><td>ab112</td><td>Dilution: 1/50</td></tr><tr><td>Triton X100</td><td>Sigma-aldrich</td><td>X100</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-aldrich</td><td>P416</td><td></td></tr></tbody></table>

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.

Using the procedure described here and summarized in Figure 1, we were able to stain and optically clear human and mouse white adipose tissue as presented in Figure 2A and Figure 2B, respectively. The cleared tissue was transferred to the metallic imaging chamber to perform confocal imaging (Figure 3A). The clearing drastically improved the depth of the tissue images that we were able to acquire (<stron...

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Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

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 ...

exploring-adipose-tissue-structure-methylsalicylate-clearing-3d

Research

JoVE Journal

Biology

7.4K Views.  Inserm UMR1065, C3M, Team "Cellular and Molecular Pathophysiology of Obesity", Nice, France. Dit protocol maakt analyse van driedimensionale structuur van Zeer grote mens op muis vetweefsel ballet microscopie. Het vergemakkelijken van de koppeling van pathologische disfunctie met vetweefsel structurele veranderingen. Dit clearing proces zijn zeer eenvoudig, zeer goed aangepast aan het opruimen van de mens op warme muizen vetweefsel en gebruik minder giftig oplosmiddel zoals ethanol of methyyl salicylaat in vergelijking met andere duidelijke protocollen.Begin met het onderdompe...

Video: Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

Bioengineering

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 Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

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 ...

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.

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 ...

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...

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. ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

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