Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 si.......

Representative 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.......

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
1x phosphate buffered salineCorning21-040-CV
ParaformaldehydeSigma AldrichP6148-1KG
MethanolFisher ScientificA412SK-4
Triton X-100Sigma AldrichX100-500ML
Tween 20Sigma AldrichP2287-500ML
HeparinSigma AldrichH3393-100KU
DichloromethaneSigma Aldrich270991
Hydrogen peroxide 30%Fisher Scientific325-100
Benzyl etherSigma Aldrich108014
AgaroseInvitrogen16500500
Sodium azideSigma Aldrich71289-5G
GlycineFisher ScientificBP381-1
Rabbit polyclonal anti-Tyrosine HydroxylaseMilliporeAB1521:200 dilution
Goat polyclonal anti-CD31/PECAM-1R&D SystemsAF3628Final concentration of 2 µg/mL
Rat monoclonal anti-CD68, Clone FA-11Bio-RadMCA1957Final concentration of 2 µg/mL
Donkey anti-rabbit IgG (H+L) Alexa Fluor 647Jackson ImmunoResearch711-605-152Final concentration of 5-10 µg/mL
Donkey anti-goat IgG (H+L) Alexa Fluor 568InvitrogenA11077Final concentration of 5-10 µg/mL
Donkey anti-rat IgG (H+L) Alexa Fluor 647Jackson ImmunoResearch712-605-153Final concentration of 5-10 µg/mL
Imaging chamberibidi80287
Light sheet microscopeLaVision BioTecUltramicroscope II
Imaging softwareLaVision BioTecImspector software
Microscopy visualization softwareBitplaneImaris

References

  1. Rosen, E. D., Spiegelman, B. M. What We Talk About When We Talk About Fat. Cell. 156 (1-2), 20-44 (2014).
  2. Barbatelli, G., et al. The emergence of cold-induced br....

Explore More Articles

Adipo ClearTissue ClearingThree dimensional ImagingAdipose TissueLipid RemovalTissue ArchitectureDehydrationDelipidationRehydrationMethanolDichloromethaneB1N BufferPTXWH Buffer

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved