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

Summary

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.

Abstract

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 of an entire adipose tissue.

Introduction

Until recently, adipose tissue was conceived of as an amorphous collection of fat cells. Over the past few decades, our understanding has grown more sophisticated, with fat now recognized to be a complex organ containing different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, the vasculature, and nerve projections. Interactions among these adipose-resident cells have pronounced effects on adipose tissue and organismal physiology and pathophysiology¹. Although emerging studies have unraveled important molecular mechanisms underlying certain interactions, a more comprehensive understanding requires reliable stru....

Protocol

Animal care and experimentation were performed according to procedures approved by the Institutional Animal Care and Use Committee at the Rockefeller University.

1. Tissue Preparation

1. Perform standard intracardiac perfusion with ~20 mL of 1x phosphate buffered saline (PBS) at 4 °C until the blood is completely removed from the tissue.
2. Switch the perfusate to ~20 mL of fixative solution (4% paraformaldehyde (PFA) in 1x PBS) at 4 °C until the neck and tail have significantly stiffened.
   CAUTION: PFA is toxic. Avoid contact with skin, eyes, and mucous membrane. Solutions should be made inside a fume hood.<....

Results

Adipo-Clear prepared whole fat pads can be imaged in 3D to analyze how tissue morphology and cellular interactions are affected in the lean and obese states. This method can be easily applied to analyze general adipose structure by collecting the tissue autofluorescence signal in the green channel. We have previously shown that the autofluorescence signal in adipose overlays favorably with perilipin staining, a commonly used marker to outline mature adipocytes¹². F.......

Discussion

Adipo-Clear is a straightforward and robust method for clearing adipose tissue, which can be easily performed in a regular lab setup. In comparison to other solvent-based clearing methods such as iDISCO/iDISCO+^10,11,12, Adipo-Clear is particularly optimized for clearing adipose tissue and other tissue with high fat content. The delipidation step completely removes lipids from adipose, and therefore facilitates immunolabeling thr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x phosphate buffered saline	Corning	21-040-CV
Paraformaldehyde	Sigma Aldrich	P6148-1KG
Methanol	Fisher Scientific	A412SK-4
Triton X-100	Sigma Aldrich	X100-500ML
Tween 20	Sigma Aldrich	P2287-500ML
Heparin	Sigma Aldrich	H3393-100KU
Dichloromethane	Sigma Aldrich	270991
Hydrogen peroxide 30%	Fisher Scientific	325-100
Benzyl ether	Sigma Aldrich	108014
Agarose	Invitrogen	16500500
Sodium azide	Sigma Aldrich	71289-5G
Glycine	Fisher Scientific	BP381-1
Rabbit polyclonal anti-Tyrosine Hydroxylase	Millipore	AB152	1:200 dilution
Goat polyclonal anti-CD31/PECAM-1	R&D Systems	AF3628	Final concentration of 2 µg/mL
Rat monoclonal anti-CD68, Clone FA-11	Bio-Rad	MCA1957	Final concentration of 2 µg/mL
Donkey anti-rabbit IgG (H+L) Alexa Fluor 647	Jackson ImmunoResearch	711-605-152	Final concentration of 5-10 µg/mL
Donkey anti-goat IgG (H+L) Alexa Fluor 568	Invitrogen	A11077	Final concentration of 5-10 µg/mL
Donkey anti-rat IgG (H+L) Alexa Fluor 647	Jackson ImmunoResearch	712-605-153	Final concentration of 5-10 µg/mL
Imaging chamber	ibidi	80287
Light sheet microscope	LaVision BioTec	Ultramicroscope II
Imaging software	LaVision BioTec	Imspector software
Microscopy visualization software	Bitplane	Imaris