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Abstract

Introduction

Protocol

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Biology

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

Published: July 28th, 2018

DOI:

10.3791/58271

1Laboratory of Molecular Metabolism, The Rockefeller University, 2Laboratory of Brain Development and Repair, The Rockefeller University, 3Laboratory of Molecular Genetics, The Rockefeller University

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 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.

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....

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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 si.......

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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.......

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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.......

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We thank Christina Pyrgaki, Tao Tong, and Alison North from the Bioimaging Resource Center at the Rockefeller University for assistance and support. We also thank Xiphias Ge Zhu for movie editing. This work was supported by the Human Frontier Science Program Organization (PC).

....

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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

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