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).

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
<ol>
	<li>Perform standard intracardiac perfusion with ~20 mL of 1x phosphate buffered saline (PBS) at 4 &#176;C until the blood is completely removed from the tissue.</li>
	<li>Switch the perfusate to ~20 mL of fixative solution (4% paraformaldehyde (PFA) in 1x PBS) at 4 &#176;C until the neck and tail have si...

<ol>
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J., Tupone, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+Neural+Regulation+of+Brown+Adipose+Tissue+Thermogenesis+and+Energy+Expenditure.">Central Neural Regulation of Brown Adipose Tissue Thermogenesis and Energy Expenditure.</a> Cell Metabolism. 19 (5), 741-756 (2014).</li><li>Xue, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-Independent+Angiogenesis+in+Adipose+Tissues+during+Cold+Acclimation.">Hypoxia-Independent Angiogenesis in Adipose Tissues during Cold Acclimation.</a> Cell Metabolism. 9 (1), 99-109 (2009).</li><li>Shimizu, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+rarefaction+mediates+whitening+of+brown+fat+in+obesity.">Vascular rarefaction mediates whitening of brown fat in obesity.</a> The Journal of Clinical Investigation. 124 (5), 2099-2112 (2014).</li><li>Abe, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+reconstruction+of+brain+section+images+for+creating+axonal+projection+maps+in+marmosets.">3D reconstruction of brain section images for creating axonal projection maps in marmosets.</a> Journal of Neuroscience Methods. 286, 102-113 (2017).</li><li>Richardson, D. S., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+Tissue+Clearing.">Clarifying Tissue Clearing.</a> Cell. 162 (2), 246-257 (2015).</li><li>Renier, N., Wu, Z., Simon, D. J., Yang, J., Ariel, P., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iDISCO:+A+Simple,+Rapid+Method+to+Immunolabel+Large+Tissue+Samples+for+Volume+Imaging.">iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging.</a> Cell. 159 (4), 896-910 (2014).</li><li>Renier, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+Brain+Activity+by+Automated+Volume+Analysis+of+Immediate+Early+Genes.">Mapping of Brain Activity by Automated Volume Analysis of Immediate Early Genes.</a> Cell. 165 (7), 1789-1802 (2016).</li><li>Chi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Adipose+Tissue+Imaging+Reveals+Regional+Variation+in+Beige+Fat+Biogenesis+and+PRDM16-Dependent+Sympathetic+Neurite+Density.">Three-Dimensional Adipose Tissue Imaging Reveals Regional Variation in Beige Fat Biogenesis and PRDM16-Dependent Sympathetic Neurite Density.</a> Cell Metabolism. 27 (1), 226-236 (2018).</li><li>Khan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Dysregulation+and+Adipose+Tissue+Fibrosis:+Role+of+Collagen+VI.">Metabolic Dysregulation and Adipose Tissue Fibrosis: Role of Collagen VI.</a> Molecular and Cellular Biology. 29 (6), 1575-1591 (2009).</li><li>Croce, A. C., Bottiroli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autofluorescence+Spectroscopy+and+Imaging:+A+Tool+for+Biomedical+Research+and+Diagnosis.">Autofluorescence Spectroscopy and Imaging: A Tool for Biomedical Research and Diagnosis.</a> European Journal of Histochemistry EJH. 58 (4), (2014).</li><li>Oh, D. Y., Morinaga, H., Talukdar, S., Bae, E. J., Olefsky, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Macrophage+Migration+Into+Adipose+Tissue+in+Obese+Mice.">Increased Macrophage Migration Into Adipose Tissue in Obese Mice.</a> Diabetes. 61 (2), 346-354 (2012).</li><li>Cinti, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipocyte+death+defines+macrophage+localization+and+function+in+adipose+tissue+of+obese+mice+and+humans.">Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans.</a> Journal of Lipid Research. 46 (11), 2347-2355 (2005).</li><li>Wang, W., Seale, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+brown+and+beige+fat+development.">Control of brown and beige fat development.</a> Nature Reviews Molecular Cell Biology. 17 (11), 691-702 (2016).</li></ol>

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...

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 structural profiling of the entire tissue in three dimensions (3D).
Our current knowledge of adipose tissue morphology is largely based on histological analysis of thin sections (5 &#956;m) with relatively high-magnification imaging (more than 10X)2,3. However, this approach has several significant limitations. First, intricate filamentous structures such as sympathetic nerves and the vasculature, which are known to play important roles in adipose function4,5,6,7, are difficult to evaluate through thin sections. Second, due to its seemingly amorphous shape and the lack of representative structural units to focus on, it is difficult to appreciate adipose tissue structures based only on section staining. Third, adipose tissue has a very high lipid content, creating challenges in obtaining consistent serial sections that are suitable for 3D anatomical reconstruction, a conventional method used to study whole brain morphology8. Given these factors, there is a great need for a whole-mount approach that can provide 3D visualization of an entire adipose depot while still achieving cellular resolution.
3D volumetric imaging of an entire organ is challenging due to the obscuring effects of light scatter. A major source of light scatter in biological tissues comes from lipid-aqueous interfaces. Although the efforts to eliminate scatter by removing lipids have been ongoing for over a century, there have been a large number of recent innovations9. One such newly developed tissue-clearing method is immunolabeling-enabled 3D imaging of solvent-cleared organs (iDISCO/iDISCO+)10,11. However, adipose tissue presents a particular challenge given its high level of lipids, and therefore, additional modifications to the iDISCO/iDISCO+ protocol are required to fully extract the lipids while protecting the tissue from collapsing. The modified protocol we have developed, now called Adipo-Clear, employs methanol/dichloromethane-based delipidation of adipose tissue to achieve optimal transparency suitable for high-resolution volumetric imaging12. Because the delipidation step largely quenches endogenously expressed fluorescent proteins such as GFP and RFP, visualization of such proteins must be achieved by immunolabeling. Overall, this simple and robust protocol can be applied to study tissue-level organization of adipose-resident cells, lineage tracing of adipocyte progenitor cells, and adipose morphogenesis during development.

<table>
	<tbody>
		<tr>
			<td>1x phosphate buffered saline</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A412SK-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>P2287-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma Aldrich</td>
			<td>H3393-100KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sigma Aldrich</td>
			<td>270991</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide 30%</td>
			<td>Fisher Scientific</td>
			<td>325-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyl ether</td>
			<td>Sigma Aldrich</td>
			<td>108014</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Invitrogen</td>
			<td>16500500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma Aldrich</td>
			<td>71289-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher Scientific</td>
			<td>BP381-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-Tyrosine Hydroxylase</td>
			<td>Millipore</td>
			<td>AB152</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Goat polyclonal anti-CD31/PECAM-1</td>
			<td>R&#38;D Systems</td>
			<td>AF3628</td>
			<td>Final concentration of 2 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Rat monoclonal anti-CD68, Clone FA-11</td>
			<td>Bio-Rad</td>
			<td>MCA1957</td>
			<td>Final concentration of 2 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit IgG (H+L) Alexa Fluor 647</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-605-152</td>
			<td>Final concentration of 5-10 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Donkey anti-goat IgG (H+L) Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A11077</td>
			<td>Final concentration of 5-10 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Donkey anti-rat IgG (H+L) Alexa Fluor 647</td>
			<td>Jackson ImmunoResearch</td>
			<td>712-605-153</td>
			<td>Final concentration of 5-10 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Imaging chamber</td>
			<td>ibidi</td>
			<td>80287</td>
			<td></td>
		</tr>
		<tr>
			<td>Light sheet microscope</td>
			<td>LaVision BioTec</td>
			<td>Ultramicroscope II</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>LaVision BioTec</td>
			<td>Imspector software</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopy visualization software</td>
			<td>Bitplane</td>
			<td>Imaris</td>
			<td></td>
		</tr>
	</tbody>
</table>

adipo-clear: a tissue clearing method for three-dimensional imaging of adipose tissue

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&#160;of an entire adipose tissue.

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...

Watch this Scientific Journal Video about Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue at JoVE.com

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

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 ...

This method can help answer key questions in the field of Adipose Tissue Biology, such as how different components of Adipose Tissue interact in an entire tissue. The main advantage of this technique is that it fully removes the excess lipids stored in Adipose Tissue while maintaining it's three-dimensional architecture. While this method can provide insight into those three-dimensional organization of Adipose Tissue it can also be generally applied to other tissues with high lipid content such as memory gland and lymph node.Visual demonstration of this method is critical as the delipidation and the clearing steps require sufficient incubation times, which can be difficult to determine. Begin by using blunt ended forceps to dissect the fat pad of interest from a freshly perfused animal model. Carefully remove the entire fad pad without pinching or squeezing the tissue or contamination the dissected tissue with fur.Place the fat pad in approximately 10 milliliters of fixation solution in a 15 milliliter conical tube and post fix at four degrees celsius overnight. The next day after washing the sample with PBS, gently remove any debris from the sample under a dissection microscope. Hair, lint, or other debris attached to the sample will cause shadows during imaging.Transfer the clean sample to a polypropylene tube containing ice cold, 20 percent methanol B1N buffer. Place the tube containing the sample horizontally on an orbital shaker, set it 100 RPM, and shake for 30 minutes to 1 hour depending on the size of the sample. Ensure the samples can move freely inside the tube.Continue dehydrating the samples through a graded series of 40 percent, 60 percent, and 80 percent methanol in B1N buffer followed by 100 percent methanol. Minimize the carryover solution. Following dehydration, replace the 100 percent methanol solution with 100 percent dichloromethane or DCM to delipidate the sample.Repeat the wash twice following the incubation times listed in table 2. The sample should sink to the bottom of the tube at the end of the second and third DCM washes. If not, extend the incubation time to ensure full delipidation.After delipidation, wash twice with 100 percent methanol for the time indicated in table 2. Rehydrate the sample in a reversed methanol B1N buffer series for the times indicated in table 2. After rehydration, wash the sample with PTXWH buffer for 2 hours at room temperature.The sample is now ready for immunostaining or could be stored in PTXWH buffer at 4 degrees celsius for up to 1 year. First, dilute the primary antibody in PTXWH buffer to the recommended concentration. Then, centrifuge the diluted antibody solution at 20 thousand G for 10 minutes to prevent introducing antibody precipitates or aggregations.It is important to choose proper primary antibodies that are compatible with methanol and DCM treatment. New antibodies should be validated with small pieces of tissue or with methanol treated tissue sections. incubate the delipidated sample with the primary antibody solution for 3 to 5 days depending on the size of the sample.After the primary incubation wash the sample with several changes of PTXWH buffer. The next day, dilute the secondary antibody in PTXWH buffer to the recommended concentration. Centrifuge the diluted antibody solution as before.Incubate the sample with the secondary antibody solution for 3 to 5 days depending on the size of the sample. Again, wash the sample with PTXWH buffer in a series of incubation steps as before. The next day, wash the sample with 1XPBS for 5 minutes, 10 minutes, then 30 minutes.Embed Adipose Tissue in agarose to facilitate sample mounting for light sheet microscopy. First, prepare the embedding solution with 1 percent agarose in 1XPBS. Cool the embedding solution to around 40 degrees celsius to avoid exposing the sample to excessive heat.While avoiding liquid carryover, place the cleaned tissue sample into a mold and arrange it into the desired position. Then gently pour the embedding solution over the sample while avoiding air bubbles. Any air bubbles should be moved to the periphery before the agarose solidifies.Once the agarose has fully solidified, cut out a block containing the sample. Transfer the sample to 25 percent methanol in water, protect from light, and incubate with shaking for the time shown in table 2. Continue dehydrating the sample through a methanol gradient.Incubate the sample in 100 percent DCM for 1 hour with shaking. DCM Washed samples dry out easily in the air, which would affect the clearness of sample and result in blurry images. It is important to transfer samples quickly into fresh solution to prevent desiccation.The sample should sink to the bottom of the tube at the end of each DCM wash. Incubate the sample in Dibenzyl ether or DBE overnight with mild shaking to achieve refractive index matching. The sample will eventually become transparent, invisible light.The next day, after incubating the sample with fresh DBE with mild shaking for 2 hours, store the sample at room temperature in the dark or proceed directly to imaging. To image the sample with a light sheet microscope that is compatible with DBE, mount the agarose embedded sample onto a holder that can prevent movement during imaging. To image the sample with an inverted confocal or two-photon microscope place the agarose embedded sample into a chamber slide with a glass bottom.Scanning a posterior subcutantious fat pad using a light sheet microscope with low magnification shows the lobular organization of Adipocytes. More detailed information such as the size of the Adipocytes can be revealed by zooming into the regions of interest with higher magnification. A posterior subcutantious fat pad stained with Tyrosine Hydroxylase, a marker for the sympathetic nervous system was imaged by light sheet florescence microscopy.TH staining revealed structures that appear as large nerve bundles as indicated by the arrow here. Blood vessel innervation as well as dense terminal arborization in the tissue Parenchyma are also revealed by TH staining. Using platelet endothelial cell adhesion molecule or PCAM1 also known as CD31 as a marker to label blood vessels, it is observed that all Adipocytes are in contact with the capillaries throughout the whole tissue supporting the high demand for efficient nutrient and oxygen exchange in Adipose.After its development, this technique paved the way for researchers in the field of Adipose Biology to explore additional questions such as early tissue morphogenesis and fate mapping of Adipocyte Progenitors. It can also be applied to study the histology of human Adipose Tissue. Don't forget that working with paraformaldehyde, methanol, dichloromethane and Dibenzyl ether can be extremely hazardous and precautions such as using a fume hood and personal protective equipment should always be taken while performing this procedure.

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26.3K Views.  The Rockefeller University. This method can help answer key questions in the field of Adipose Tissue Biology, such as how different components of Adipose Tissue interact in an entire tissue. The main advantage of this technique is that it fully removes the excess lipids stored in Adipose Tissue while maintaining it's three-dimensional architecture. While this method can provide insight into those three-dimensional organization of Adipose Tissue it can also be generally applied to other tissues with high lipid content su...