Name: exploring adipose tissue structure by methylsalicylate clearing and 3d imaging
Uploaded: 2020-08-19T15:00:00
Description: Watch this Scientific Journal Video about Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging at JoVE.com

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 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 ...

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