Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Summary

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.

Abstract

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.

Introduction

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 homeostasis^1,2. During the feeding period, the adipocytes (i.e., the main cells of the adipose tissue) store the excess of glucose and lipids provided by a meal into triglycerides. During fasting, the adipocytes break down the tr....

Protocol

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

1. 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.
2. Shake the plastic tube at room temperature on a rolling plate for 1 h.
3. Leave the plastic tube at 4 °C on a rolling plate overnight, to complete the fixation.
   NOTE: This protocol is....

Results

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 (

Discussion

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 cytometry^22,23, and transcriptomics^24,

Materials

List of materials used in this article

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