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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....
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
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.......
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.......
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).
....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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