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 significantly stiffened. 
	CAUTION: PFA is toxic. Avoid contact with skin, eyes, and mucous membrane. Solutions should be made inside a fume hood.</li>
	<li>Dissect the fat pads of interest carefully to remove the entire fat pad without damaging it. Use blunt-ended forceps to avoid pinching or squeezing the tissue. Avoid contaminating the dissected tissue with any fur.</li>
	<li>Post-fix the tissue in 4% PFA in 1x PBS overnight at 4 &#176;C. For each fat pad, post-fix with ~10 mL of fixation solution in a 15 mL conical tube.</li>
	<li>Wash the tissue 3 times (1 h each) with 1x PBS at room temperature (RT).</li>
	<li>Store the tissue for short-term (up to 2 weeks) in 1x PBS at 4 &#176;C and protected from light. For long-term storage (e.g., 1-2 years), switch the buffer to 1x PBS with 0.05% sodium azide (as a preservative). 
	CAUTION: Sodium azide is highly toxic when ingested orally or absorbed through the skin. Concentrated sodium azide solutions (at 5% or greater) should be handled under a fume hood.</li>
</ol>
2. Delipidation and Permeabilization
Timing: 1-2 days
<ol>
	<li>Prepare 20%, 40%, 60%, and 80% methanol gradient with B1n buffer (Table 1) (v/v), e.g., for 20% methanol/B1n buffer, mix 20 mL of methanol and 80 mL of B1n buffer. Store all buffers at 4 &#176;C. Glycine crystals will precipitate from 60% and 80% methanol/B1n buffers due to saturation. Avoid removing the crystals; use the liquid solution for the following washes. 
	CAUTION: Methanol is volatile, irritant, and flammable. Avoid skin or eye contact.</li>
	<li>Gently remove the hair from the sample under a dissection microscope. Any hair, lint, or debris attached to the sample will cause shadows during imaging. Transfer the cleaned sample into a new tube.</li>
	<li>Perform all following steps on ice or at 4 &#176;C unless otherwise indicated. For small samples (e.g., posterior subcutaneous/perigonadal fat pads from young lean mice), use 2 mL microcentrifuge tubes with 1.6 mL of the solution. For large samples or samples with high lipid content (e.g., fat pads from obese mice), use 5 mL tubes with 4 mL of the solution.
	<ol>
		<li>Place the tubes containing the samples horizontally on an orbital shaker set at ~ 100 rpm. Make sure the samples can move freely inside the tube.</li>
	</ol>
	</li>
	<li>Dehydrate the sample in a graded series of 20%, 40%, 60%, 80%, and 100% methanol/B1n buffer for the indicated time (Table 2). Minimize the carry-over solution.</li>
	<li>Delipidate the sample with 100% dichloromethane (DCM) (3 times), each with the incubation time indicated 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 (e.g., extend from 30 min to 1-2 h). 
	CAUTION: DCM should be handled inside a fume hood. Use glass containers for storage and microcentrifuge tubes that are made from polypropylene for incubations. The microcentrifuge tubes should not be used for long-term storage of DCM. Petri dishes and serological pipettes that are made from polystyrene are not compatible with DCM. DCM is highly volatile. Transfer the sample into fresh solution quickly to prevent desiccation.</li>
	<li>Wash twice with 100% methanol for the indicated time (Table 2).</li>
	<li>Optional: If the sample is not well-perfused, the color of hemoglobin from the remaining red blood cells might cause strong autofluorescence. Bleach with 5% H2O2 in methanol (1 volume of 30% H2O2 to 5 volumes of methanol) overnight at 4 &#176;C.</li>
	<li>Rehydrate the sample in a reversed methanol/B1n buffer series: 80%, 60%, 40%, 20% methanol/B1n, and 100% B1n buffer (2 times) for the indicated time (Table 2).</li>
	<li>Wash the sample with PTxwH buffer (Table 1) for 2 h at RT.</li>
	<li>Store the delipidated sample in PTxwH buffer at 4&#176; C (up to 1 year) or proceed immediately to the immunostaining step.</li>
</ol>
3. Whole Mount Immunostaining
Timing: 8-10 days
Note: All of the following steps should be carried out at RT unless noted otherwise, with shaking and protection from light. For small samples, use 2 mL microcentrifuge tubes with 1.6 mL of solution. For large samples, use 5 mL tubes with 4 mL of solution. It is recommended to first validate the antibodies on small pieces of the tissue or methanol-treated tissue sections.
<ol>
	<li>Dilute the primary antibody in PTxwH buffer to the recommended concentration. Centrifuge the diluted antibody solution at ~20,000 x g for 10 min to prevent introducing antibody precipitates or aggregations.</li>
	<li>Incubate the delipidated sample with the primary antibody solution for the indicated time (Table 2).</li>
	<li>Wash the sample with PTxwH buffer in a series of incubation steps: 5 min, 10 min, 15 min, 20 min, 1 h, 2 h, 4 h, and overnight.</li>
	<li>Dilute the secondary antibody in PTxwH buffer to the recommended concentration. Centrifuge the diluted antibody solution at ~20,000 x g for 10 min to prevent introducing antibody precipitates or aggregations. 
	Note: To avoid high background caused by tissue autofluorescence, secondary antibodies conjugated with fluorophores that emit light in the red and far-red regions are recommended. Depending on the number of laser lines available on a microscope, up to 3-4 markers can be imaged simultaneously in one sample.</li>
	<li>Incubate the sample with the secondary antibody solution for the indicated time (Table 2).</li>
	<li>Wash sample with PTxwH buffer in a series of incubation steps: 5 min, 10 min, 15 min, 20 min, 1 h, 2 h, 4 h, and overnight.</li>
	<li>Optional: For tissue types that are fragile, fix the stained tissue with 4% PFA in 1x PBS overnight at 4 &#176;C to help preserve tissue morphology. 
	Note: This step may increase imaging background. It is not necessary to perform this step for adipose tissue.</li>
	<li>Wash the sample with 1x PBS in a series of incubation steps: 5 min, 10 min, and 30 min.</li>
	<li>Gently remove lint from the sample under a dissection microscope.</li>
</ol>
4. Tissue Clearing
Timing: 1-2 days
<ol>
	<li>Optional: Embed the sample in agarose to facilitate sample mounting for light-sheet microscopy. 
	Note: This step is strongly recommended for adipose tissue to stabilize its shape during imaging.
	<ol>
		<li>Prepare the embedding solution with 1% agarose in 1x PBS (w/v). Cool the embedding solution to ~ 40 &#176;C to avoid exposing the sample to excessive heat.</li>
		<li>Place the cleaned sample into a mold (e.g., Petri dish or weighing boat) and arrange it into the desired position. Avoid any liquid carryover. Gently pour the embedding solution over the sample. Avoid any air bubbles.</li>
		<li>Let the agarose fully solidify at RT. Cut out a block containing the sample. 
		Note: All of the following steps including overnight incubations should be carried out at RT, with shaking and protection from light. For small samples, use 2 mL microcentrifuge tubes with 1.6 mL of solution. For large samples, use 5 mL tubes with 4 mL of solution.</li>
	</ol>
	</li>
	<li>Dehydrate the sample in methanol gradient with H2O: 25%, 50%, 75%, and 100% (3 times) for the indicated time (Table 2). 
	Note: Sample can be left overnight at the 100% methanol step.</li>
	<li>Incubate the sample in 100% DCM for 1 h with shaking (3 times). Sample should sink to the bottom of the tube at the end of each DCM wash. If not, extend the incubation time to ensure full removal of methanol. Sample can be left overnight at the 100% DCM step.</li>
	<li>Incubate the sample in dibenzyl ether (DBE) overnight with mild shaking to achieve refractive index matching. 
	Note: Sample will eventually become transparent in visible light. 
	CAUTION: DBE is hazardous. Avoid any contact with skin. Handle it inside a fume hood with double-layered gloves. Discard the gloves as soon as it is contaminated with DBE. Use glass containers for storage and microcentrifuge tubes (polypropylene) for incubations. The microcentrifuge tubes should not be used for long-term storage of DBE. Petri dishes and serological pipettes that are made from polystyrene are not compatible with DBE. Clean any spill with 100% ethanol.</li>
	<li>Incubate the sample with fresh DBE with mild shaking for 2 h. Store the sample at RT in the dark or proceed directly to imaging. 
	Note: Samples are recommended to be imaged within a month. Although certain fluorophores are more stable than others, long-term storage of the samples at this stage is not recommended.</li>
</ol>
5. Microscopy
<ol>
	<li>Imaging with a light-sheet microscope that is compatible with DBE. 
	Note: Only objectives that are approved for organic solvent-based imaging and matched with the refractive index of DBE can be used as dipping objectives in DBE-immersed imaging.
	<ol>
		<li>Mount the agarose-embedded sample onto a holder that can prevent movement during imaging. Immerse the sample into a DBE-filled chamber.</li>
		<li>Collect a z-stack covering the whole sample (e.g., 1-2X magnification) to obtain whole-tissue distribution/structure of the marker of interest.</li>
		<li>Switch to an objective with higher magnification (e.g., 4-12X magnification) to zoom in on the regions of interest to image detailed structures. 
		Note: Collagen is a major contributor to the extracellular matrix of adipose tissue, which surrounds all fat cells13. Collagen has a typical emission spectrum ranging around 400 nm to 550 nm14. Imaging the sample with the 488 laser line (green) and collecting the emitted light at a wavelength range that lies within the emission spectrum of collagen (e.g., 525-550 nm) will provide the tissue autofluorescence signal, which was previously shown to delineate fat cell contour and overall tissue architecture12.</li>
	</ol>
	</li>
	<li>Imaging with an inverted confocal or a two-photon microscope.
	<ol>
		<li>Place the agarose-embedded sample into a chamber slide with a glass bottom without touching any plastic parts (or other DBE-compatible imaging chamber that can seal). Make sure DBE does not leak out.</li>
		<li>Choose the objectives with longer working distances to achieve deeper penetration. 
		Note: Imaging should be done with an inverted confocal or two-photon microscope through the glass bottom of the chamber slide. Avoid direct contact between the sample and dipping objectives that are not suggested for DBE-based imaging.</li>
	</ol>
	</li>
</ol>

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	<li>Rosen, E. D., Spiegelman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+We+Talk+About+When+We+Talk+About+Fat.">What We Talk About When We Talk About Fat.</a> Cell. 156 (1-2), 20-44 (2014).</li><li>Barbatelli, G., 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=The+emergence+of+cold-induced+brown+adipocytes+in+mouse+white+fat+depots+is+determined+predominantly+by+white+to+brown+adipocyte+transdifferentiation.">The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation.</a> American Journal of Physiology - Endocrinology and Metabolism. 298 (6), E1244-E1253 (2010).</li><li>Wang, Q. A., Tao, C., Gupta, R. K., Scherer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+adipogenesis+during+white+adipose+tissue+development,+expansion+and+regeneration.">Tracking adipogenesis during white adipose tissue development, expansion and regeneration.</a> Nature Medicine. 19 (10), 1338-1344 (2013).</li><li>Bartness, T. J., Liu, Y., Shrestha, Y. B., Ryu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+innervation+of+white+adipose+tissue+and+the+control+of+lipolysis.">Neural innervation of white adipose tissue and the control of lipolysis.</a> Frontiers in Neuroendocrinology. 35 (4), 473-493 (2014).</li><li>Morrison, S. F., Madden, C. 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. 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The authors have nothing to disclose.

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&amp;amp;D Systems</td><td>AF3628</td><td>Final concentration of 2 &amp;micro;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 &amp;micro;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 &amp;micro;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 &amp;micro;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 &amp;micro;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 ...

adipo-clear-tissue-clearing-method-for-three-dimensional-imaging

Research

JoVE Journal

Biology

27.1K Views.  The Rockefeller University. Als gevolg van het hoge vetgehalte, heeft adipeus weefsel is uitdagend om te visualiseren met behulp van traditionele histologische methoden. Adipo-Clear is een weefsel clearing techniek waarmee robuuste labeling en hoge resolutie volumetrische fluorescerende beeldvorming van vetweefsel. Hier beschrijven we de methoden voor de bereiding van de monsters, voorbehandeling, kleuring, clearing en montage voor imaging.

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

Imaging Cleared Intact Biological Systems at a Cellular Level by 3DISCO

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact organs. Unlike traditional histological methods, where the tissue of interest is sectioned for fluorescent imaging, 3D imaging of cleared tissue allows examination of labeled cells and molecules in the entire specimen. To this end, optically opaque tissues should be rendered transparent by matching the refractory indices throughout the tissue. Subsequently, the tissue can be imaged at once using laser-scanning microscopes to obtain a complete high-resolution 3D image of the specimen. A growing list of tissue clearing protocols including 3DISCO, CLARITY, Sca/e, ClearT2, and SeeDB provide new ways for researchers to image their tissue of interest as a whole. Among them, 3DISCO is a highly reproducible and straightforward method, which can clear different types of tissues and can be utilized with various microscopy techniques. This protocol describes this straightforward procedure and presents its various applications. It also discusses the limitations and possible difficulties and how to overcome them.

To obtain high-resolution images of fluorescently labeled cells within large tissues, ideally, the biological samples should be imaged without sectioning. 3DISCO is a straightforward tissue clearing procedure based on sequential incubation with organic solvents. Upon clearing, the organs become transparent allowing an end-to-end laser scan of the specimen.

Tissue clearing and subsequent imaging of transparent organs is a powerful method to analyze fluorescently labeled cells and molecules in 3D, in intact ...

Neuroscience

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Medicine

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

Immunology and Infection

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Human SATMVECs: Isolation and Adipocyte Cross-Talk Studies

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

Author Spotlight: Insights into Cardiometabolic Diseases with Subcutaneous Adipose Tissue Microvasculature Studies 

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. ...

Three-Dimensional Adipocyte Culture as a Model to Study Cachexia-Induced White Adipose Tissue Remodeling

Cancer cachexia (CC) presents itself as a syndrome with multiple manifestations, causing a marked multi-organ metabolic imbalance. Recently, cachectic wasting has been proposed to be stimulated by several inflammatory mediators, which may disrupt the integrative physiology of adipose tissues and other tissues such as the brain and muscle. In this scenario, the tumor can survive at the host&#39;s expense. In recent clinical research, the intensity of depletion of the different fat deposits has been negatively correlated with the patient&#39;s survival outcome. Studies have also shown that various metabolic disorders can alter white adipose tissue (WAT) remodeling, especially in the early stages of cachexia development. WAT dysfunction resulting from tissue remodeling is a contributor to overall cachexia, with the main modifications in WAT consisting of morpho-functional changes, increased adipocyte lipolysis, accumulation of immune cells, reduction of adipogenesis, changes in progenitor cell population, and the increase of &#34;niches&#34; containing beige/brite cells.
To study the various facets of cachexia-induced WAT remodeling, particularly the changes&#160;progenitor cells and beige remodeling, two-dimensional (2D) culture has been the first option for in vitro studies. However, this approach does not adequately summarize WAT complexity. Improved assays for the reconstruction of functional AT ex vivo&#160;help the comprehension of physiological interactions between the distinct cell populations. This protocol describes an efficient three-dimensional (3D) printing tissue culture system based on magnetic nanoparticles. The protocol is optimized for investigating WAT remodeling induced by cachexia induced factors (CIFs). The results show that a 3D culture is an appropriate tool for studying WAT modeling ex vivo and may be useful for functional screens to identify bioactive molecules for individual adipose cell populations applications and aid the discovery of WAT-based cell anticachectic therapy.

This protocol describes a three-dimensional (3D) magnetic printing culture system that permits dissection of white adipose tissue (WAT) remodeling induced by a conditioned medium from cancer cells. Using a 3D culture system of UCP1+ adipocytes that express a green fluorescent protein (GFP) allows the study of beige adipocytes contributing to adipose tissue remodeling.

Cancer cachexia (CC) presents itself as a syndrome with multiple manifestations, causing a marked multi-organ metabolic imbalance. Recently, cachectic ...

Imaging the Gut with "CLARITY"

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving acrylamide embedding to delipidate tissue (without sectioning) and to preserve the 3-D tissue structure for immunostaining. The technique is highly relevant in imaging the dynamic gut environment where different cell types interact during homeostasis and disease states. This method optimized for the mouse gut is described here, which helps to trace cell types like epithelia, enteroendocrine, neurons, glia, and the neuronal projections into the epithelia or enteroendocrine cells that mediate microbial sensing or nutrient chemo sensing respectively. The gut tissue (1-1.5 cm) is fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 &#176;C overnight on day 1. On day 2, PFA is discarded, and the tissue is washed thrice with PBS. The tissue is hydrogel embedded to preserve its integrity by incubation in 4% hydrogel (acrylamide) solution in PBS (diluted from 30% ProtoGel) overnight at 4 &#176;C. On day 3, the tissue-hydrogel solution is incubated at 37&#160;&#176;C for 1 h to allow hydrogel polymerization. Tissue is then washed thrice gently with PBS to remove excess hydrogel. The subsequent step of delipidation (clearing) involves tissue incubation in sodium dodecyl sulfate (8% SDS in PBS) at 37 &#176;C for 2 days (days 4 &#38; 5) on a shaker at room temperature (RT). On day 6, the cleared tissue is thoroughly washed with PBS to remove SDS. Tissue can be immunostained by incubation in primary antibodies (diluted in 0.5% normal donkey serum in PBS containing 0.3% Triton X-100), overnight at 4&#176;C, and subsequent incubation in appropriate secondary Alexa Fluor antibodies for 1.5 h at RT, and nuclear staining with DAPI (1: 10000). The tissue is transferred onto a clean glass slide and mounted using VectaShield for confocal imaging.

We describe the protocol for passive CLARITY (PACT) staining of mouse intestine to enable visualization of subepithelial tissues, including neurons, glia and enteroendocrine cells (EEC) without tissue sectioning. The protocol involves hydrogel embedding of formaldehyde-fixed tissue, and subsequent delipidation using an anionic detergent to "clear" the tissue.

CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging compatible Tissue hYdrogel) has recently evolved as a valuable technique involving ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

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

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