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 roll...

<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 &#181;g/&#181;L; IV Injection: 50 &#181;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-&#946;-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...

Watch this Scientific Journal Video about Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging at JoVE.com

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 ...

These protocol allows analysis of three-dimensional structure of Very large human on mouse adipose tissue ballet microscopy. Facilitating the linkage of pathological dysfunction with adipose tissue structural changes. This clearing process are very simple, very well adapted to clearing human on warm mice adipose tissue and use less toxic solvent such as ethanol or methyl salicylate compared to other clear protocols.Begin by immersing the harvested mouse or human white adipose tissue in at least 10 milliliters of 4%power formaldehyde in PBS in a 15 milliliter plastic tube preferentially under a chemical hood. Shake the tissue on a rolling plate at room temperature for one hour before transferring the tube to a rolling plate at four degrees Celsius overnight. The next morning rinse the fixed white adipose tissue in 10 milliliters of PBS for five minutes at room temperature to remove all traces of fixative and transfer the tissue to a 15 milliliter tube containing 10 milliliters of 0.3%glycine in PBS for a one hour incubation at room temperature on an orbital shaker at 100 revolutions per minute.When the remaining free aldehyde groups have been quenched, immerse the tissue in a 15 milliliter tube containing 10 milliliters of 0.2%Triton X-100 in PBS for a two hour incubation with shaking at 37 degrees Celsius. At the end of the incubation treat the tissue with 10 milliliters of 0.2%Triton X-100 and 20%DMSO with shaking at 37 degrees Celsius overnight. The next morning immerse the tissue in a 15 milliliter plastic tube containing 10 milliliters of 0.1%Tween 20, 0.1%Triton X-100, 0.1%deoxycholate and 20%DMSO in PBS for an at least 24 hour incubation at 37 degrees Celsius with shaking.At the end of the incubation, rinse the tissue with 10 milliliters of 0.2%Triton X-100 in PBS on an orbital shaker at 100 revolutions per minute for one hour. Followed by immersion in 15 milliliters of 0.2%Triton X-100, 10%DMSO and 3%BSA in PBS for 12 hours at 37 degrees Celsius with shaking. For staining of the white adipose tissue.Transfer the tissue into 300 microliters of 0.2%Triton X-100, 10%DMSO and 3%BSA in PBS, supplemented with the primary antibodies of interest in a 1.5 milliliter micro tube protect it from light with aluminum foil and place the tube into a 50 milliliter Falcon tube. Place the tube into the orbital shaker for a two day incubation at 37 degrees Celsius with shaking. At the end of the incubation rinse the tissue two times in 10 milliliters of 0.2%Triton X-100, 10%DMSO and 3%BSA in PBS for five hours at 37 degrees Celsius with shaking and protection from light.After the second rinse wash the tissue in 10 milliliters of fresh 0.2%Triton X-100, 10%DMSO and 3%BSA and PBS for 16 to 48 hours at 37 degrees Celsius with shaking, then transfer the tissue to a 1.5 milliliter tube containing 300 microliters of 0.2%Triton X-100, 10%DMSO and 3%BSA in PBS, supplemented with the appropriate secondary antibodies and protect the tube from light using aluminum foil. After a two day incubation at 37 degrees Celsius on a shaker, wash the tissue two times in 10 milliliters of 0.2%Triton X-100, 10%DMSO and 3%BSA in PBS for five hours at 37 degrees Celsius with shaking protected from light. After the second wash, rinse the tissue into a tube containing 10 milliliters of fresh 0.2%Triton X-100, 10%DMSO and 3%BSA in PBS for 16 to 48 hours at 37 degrees Celsius with shaking and light protection.Followed by a two hour wash in 10 milliliters of PBS alone at 37 degrees Celsius with shaking and light protection. For white adipose tissue clearing at the end of the PBS wash immerse the tissue and 10 milliliters of an ascending sending ethanol concentration series for a two hour incubation per concentration at room temperature with shaking protected from light as indicated. The next morning immerse the tissue in a 20 milliliter glass bottle with a plastic cap containing five milliliters of methyl salicylate in a fume hood.The white adipose tissue is still clearly visible at this stage. Shake the container protect it from light at room temperature for at least two hours. The white adipose tissue becomes completely transplant.For 3D confocal imaging of the stained and cleared white adipose tissue mount a metallic imaging chamber equipped with a glass bottom and in a fume hood transfer the tissue to the chamber. Fill the chamber with fresh methyl salicylate. Finalize the mountaging of the chamber by adding a small cover slip to stabilize the tissue and a large cover slip to close the chamber.Place the chamber onto an inverted confocal microscope stage for tissue imaging at low magnification to generate a few cubic centimeter 3D maps of the whole adipose tissue sample. After 3D map generation, use a 20X long distance air objective that provides a good ratio between the resolution and depth to select several areas for tissue sampling at a higher magnification. For cell segmentation convert 3D stacks onto the software format to free up memory space.Before opening the cell module of the software. Change the cell detection setting to plasma membrane staining and select the fluorescent channel of the marker used to delineate the cell periphery. Then set up the thresholds, provide a range of the expected cell sizes and run the segmentation.The affects of clearing on human and mouse white adipose tissue can be observed with the naked eye. Clearing also drastically improves the image depth of the tissue that is able to be acquired. And facilitates whole tissue 3D imaging and 3D map generation out of 4X Magnification.3D mapping further enables the selection of different areas of interest to be acquired at a 20X magnification to a depth of two millimeters. Specific staining of lipid droplets and adipocytes can be achieved using perilipin and anti-GLUT4 antibodies respectively. Blood vessels can be detected using either CD31 antibody or intravenous injection of Lectin DyLight 649 shortly before sacrifice.Macrophages and T-cells can be visualized using anti CD301 phycoerythrin antibody and anti TCR beta Pacific blue antibody. The peripheral nerve network can be detected using anti Tyrosine Hydroxylase antibody. Using these labelings the mean size and size distribution of the adipocytes and the blood vessel network density within the adipose tissue can then be determined.It's crucial to follow occupation time as demonstrated because poor sample preparation will not result in a good image quality. This protocol can be combined with advanced imaging system or can be coupled to post-processing image analysis freeware.

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

Research

JoVE Journal

Biology

7.2K Görüntüleme.  Inserm UMR1065, C3M, Team "Cellular and Molecular Pathophysiology of Obesity", Nice, France. Bu protokol fare yağ dokusu bale mikroskopisi üzerinde çok büyük insan üç boyutlu yapısının analizini sağlar. Patolojik disfonksiyonun yağ dokusu yapısal değişiklikleri ile bağlantısını kolaylaştırmak. Bu temizleme işlemi çok basit, çok iyi sıcak fareler yağ dokusu üzerinde insan temizleme ve diğer açık protokollere göre etanol veya metil salisilat gibi daha az toksik çözücü kullanmak adapte.Hasat edilen fareyi veya insan beyaz yağ dokusunu pbs'de %4...