Name: identification and dissection of diverse mouse adipose depots
Uploaded: 2019-07-11T12:30:00
Description: Watch this Scientific Journal Video about Identification and Dissection of Diverse Mouse Adipose Depots at JoVE.com

O.A.M. is supported by NIH grants DK062876 and DK092759; D.P.B. is supported by the University of Michigan Medical Scientist Training Program (T32GM007863), University of Michigan Training Program in Organogenesis (T32HD007605), University of Michigan Rackham Merit Fellowship, and Tylenol Future Care Fellowship.

All animal procedures are performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan.
1. Euthanization
NOTE: For the purpose of this video protocol, 4 to 6 month-old C57BL/6J mice are used.
<ol>
	<li>Place the mouse in an isoflurane vaporizer chamber and adjust the isoflurane flow rate to 5% or greater. Continue isoflurane exposure until one minute after breathing stops. Then remove the mou...

<ol>
	<li>Cinti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adipose+organ+at+a+glance.">The adipose organ at a glance.</a> Disease Models &amp; Mechanisms. 5 (5), 588-594 (2012).</li><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>Sanchez-Gurmaches, J., Guertin, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipocyte+lineages:+tracing+back+the+origins+of+fat.">Adipocyte lineages: tracing back the origins of fat.</a> Biochimica et Biophysica Acta. 1842 (3), 340-351 (2014).</li><li>Bagchi, D. P., Forss, I., Mandrup, S., MacDougald, O. 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M., 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=Brown+and+Beige+Fat:+Physiological+Roles+beyond+Heat+Generation.">Brown and Beige Fat: Physiological Roles beyond Heat Generation.</a> Cell Metabolism. 22 (4), 546-559 (2015).</li><li>Frontini, A., Cinti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+development+of+brown+adipocytes+in+the+murine+and+human+adipose+organ.">Distribution and development of brown adipocytes in the murine and human adipose organ.</a> Cell Metabolism. 11 (4), 253-256 (2010).</li><li>Zhang, F., 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=An+Adipose+Tissue+Atlas:+An+Image-Guided+Identification+of+Human-like+BAT+and+Beige+Depots+in+Rodents.">An Adipose Tissue Atlas: An Image-Guided Identification of Human-like BAT and Beige Depots in Rodents.</a> Cell Metabolism. 27, 252-262 (2018).</li><li>Sanchez-Gurmaches, J., Guertin, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipocytes+arise+from+multiple+lineages+that+are+heterogeneously+and+dynamically+distributed.">Adipocytes arise from multiple lineages that are heterogeneously and dynamically distributed.</a> Nature Communications. 5, 4099 (2014).</li><li>Li, Z., Hardij, J., Bagchi, D. P., Scheller, E. L., MacDougald, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+regulation,+metabolism+and+function+of+bone+marrow+adipose+tissues.">Development, regulation, metabolism and function of bone marrow adipose tissues.</a> Bone. 110, 134-140 (2018).</li><li>Scheller, E. L., Cawthorn, W. P., Burr, A. A., Horowitz, M. C., MacDougald, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marrow+Adipose+Tissue:+Trimming+the+Fat.">Marrow Adipose Tissue: Trimming the Fat.</a> Trends in Endocrinology &amp; Metabolism. 27 (6), 392-403 (2016).</li><li>Alexander, C. M., 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=Dermal+white+adipose+tissue:+a+new+component+of+the+thermogenic+response.">Dermal white adipose tissue: a new component of the thermogenic response.</a> The Journal of Lipid Research. 56 (11), 2061-2069 (2015).</li><li>Kruglikov, I. L., 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=Dermal+Adipocytes:+From+Irrelevance+to+Metabolic+Targets?.">Dermal Adipocytes: From Irrelevance to Metabolic Targets?.</a> Trends in Endocrinology &amp; Metabolism. 27 (1), 1-10 (2016).</li><li>Iacobellis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+and+systemic+effects+of+the+multifaceted+epicardial+adipose+tissue+depot.">Local and systemic effects of the multifaceted epicardial adipose tissue depot.</a> Nature Reviews Endocrinology. 11 (6), 363-371 (2015).</li><li>Addison, O., Marcus, R. L., LaStayo, P. C., Ryan, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermuscular+Fat:+A+Review+of+the+Consequences+and+Causes.">Intermuscular Fat: A Review of the Consequences and Causes.</a> International Journal of Endocrinology. 2014, 1-11 (2014).</li><li>Pond, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+and+the+immune+system.">Adipose tissue and the immune system.</a> Prostaglandins, Leukotrienes, and Essential Fatty Acids. 73 (1), 17-30 (2005).</li><li>Kloppenburg, A. I. -. F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+emerging+player+in+knee+osteoarthritis:+the+infrapatellar+fat+pad.">An emerging player in knee osteoarthritis: the infrapatellar fat pad.</a> Arthritis Research &amp; Therapy. 15 (225), 1-9 (2013).</li><li>Mann, A., Thompson, A., Robbins, N., Blomkalns, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization,+Identification,+and+Excision+of+Murine+Adipose+Depots.">Localization, Identification, and Excision of Murine Adipose Depots.</a> Journal of Visualized Experiments. (94), e52174 (2014).</li><li>Casteilla, L., Cousin, B., Calise, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+an+adipose+tissue+depot+for+sampling:+factors+in+selection+and+depot+specificity.">Choosing an adipose tissue depot for sampling: factors in selection and depot specificity.</a> Methods in Molecular Biology. 155, 1-22 (2008).</li><li>de Jong, J. M., Larsson, O., Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stringent+validation+of+mouse+adipose+tissue+identity+markers.">A stringent validation of mouse adipose tissue identity markers.</a> American Journal of Physiology-Endocrinology and Metabolism. 308 (12), E1085-E1105 (2015).</li><li>Cinti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adipose+organ.">The adipose organ.</a> Prostaglandins, Leukotrienes, and Essential Fatty Acids. 73 (1), 9-15 (2005).</li><li>Parlee, S. D., Lentz, S. I., Mori, H., MacDougald, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+size+and+number+of+adipocytes+in+adipose+tissue.">Quantifying size and number of adipocytes in adipose tissue.</a> Methods in Enzymology. 537, 93-122 (2014).</li><li>Scheller, E. L., 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=Region-specific+variation+in+the+properties+of+skeletal+adipocytes+reveals+regulated+and+constitutive+marrow+adipose+tissues.">Region-specific variation in the properties of skeletal adipocytes reveals regulated and constitutive marrow adipose tissues.</a> Nature Communications. 6, 7808 (2015).</li><li>Scheller, E. L., 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=Use+of+osmium+tetroxide+staining+with+microcomputerized+tomography+to+visualize+and+quantify+bone+marrow+adipose+tissue+in+vivo.">Use of osmium tetroxide staining with microcomputerized tomography to visualize and quantify bone marrow adipose tissue in vivo.</a> Methods in Enzymology. 537, 123-139 (2014).</li><li>Lukjanenko, L., Brachat, S., Pierrel, E., Lach-Trifilieff, E., Feige, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+profiling+reveals+that+transient+adipogenic+activation+is+a+hallmark+of+mouse+models+of+skeletal+muscle+regeneration.">Genomic profiling reveals that transient adipogenic activation is a hallmark of mouse models of skeletal muscle regeneration.</a> PLoS One. 8 (8), e71084 (2013).</li><li>Pagano, A. F., 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=Muscle+Regeneration+with+Intermuscular+Adipose+Tissue+(IMAT)+Accumulation+Is+Modulated+by+Mechanical+Constraints.">Muscle Regeneration with Intermuscular Adipose Tissue (IMAT) Accumulation Is Modulated by Mechanical Constraints.</a> PLoS One. 10 (12), e0144230 (2015).</li><li>Khan, I. M., 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=Intermuscular+and+perimuscular+fat+expansion+in+obesity+correlates+with+skeletal+muscle+T+cell+and+macrophage+infiltration+and+insulin+resistance.">Intermuscular and perimuscular fat expansion in obesity correlates with skeletal muscle T cell and macrophage infiltration and insulin resistance.</a> International Journal of Obesity. 39 (11), 1607-1618 (2015).</li><li>Sulston, R. 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As the importance of the diverse molecular and functional characteristics of discrete adipocyte clusters is increasingly recognized, it is crucial that investigators within the field uniformly identify and excise adipose depots for further analyses. To date, few protocols exist for standardized localization and isolation of the wide range of mouse adipose depots. Previously published methods are focused primarily on one or two depots and lack the details necessary for uniform identification and excision by different inve...

Adipose tissues play critical roles in whole-body homeostasis, including storage and release of energy in response to local and global needs, thermoregulation, and secretion of adipokines to regulate energy balance, metabolism and immune responses1,2. Adipocytes are distributed throughout the body in discrete depots, and in some cases serve specialized roles within their microenvironments3,4,5. Historically, the study of adipose tissue has centered on white adipose tissue (WAT), and its role in maintaining energy homeostasis. Most adipocytes are distributed throughout the body in subcutaneous and visceral WAT depots. The characteristics of these depots are important for differential susceptibility to metabolic diseases. Subcutaneous adipocytes, located beneath the skin, have been associated with protective metabolic effects5. Visceral adipocytes, which surround the vital organs and are contained within the gonadal, perirenal, retroperitoneal, omental and pericardial depots, are commonly linked to metabolic disorders, including type 2 diabetes and cardiovascular disease2. Brown adipose tissues (BAT) have also been studied extensively. Brown and brown-like adipocytes express uncoupling protein 1 (UCP1) and play important roles in adaptive thermogenesis and glucose homeostasis6,7. Classical brown adipocytes are contained in the interscapular BAT depot8. Clusters of brown adipocytes are also found in other locations, including supraclavicular, infra/subscapular, cervical, paravertebral and periaortic depots8,9.
In addition to their location in major WAT and BAT depots, adipocytes exist in discrete niches throughout the body4, where they may perform specialized functions within their respective microenvironments. For example, bone marrow adipose tissue (BMAT) serves as a lipid reservoir, is a major source of circulating adiponectin, and closely interacts with osteoblasts, osteoclasts, and hematopoietic cells10,11. Dermal adipocytes contribute to widespread processes, including wound healing, immune response, thermoregulation, and hair follicle growth12,13. Further, epicardial adipocytes may produce several adipokines and chemokines that exert local and systemic effects on the development and progression of coronary artery disease14. Expansion of inter/intramuscular WAT has been positively correlated with increased adiposity, systemic insulin resistance, and decreased muscular strength and mobility15. In addition, popliteal adipocytes serve as a lipid reservoir for lymphatic expansion during infection16. While the specific roles of different articular depots are generally unknown, the Hoffa depot (infrapatellar) within the knee is now thought to contribute to pathologies, including anterior knee pain and osteoarthritis17.
Whereas regional differences in adipocyte character and function are under intense study, the field is currently limited by the lack of a standardized protocol for the identification and dissection of diverse mouse depots. Previously published methods have typically described the isolation of one or two specific depots and lack the level of detail required for uniform excision18,19. The protocol described in this manuscript provides a comprehensive guide for the specific anatomic locations and isolation steps of many different mouse adipose depots. Although WAT depots are the primary focus of this manuscript, the excision of interscapular BAT is also described in detail. Adipose tissues excised using this protocol can be used for a wide variety of experimental endpoints, including&#160;explant studies, histology, and gene expression analyses.
The goal of this manuscript is to provide investigators with a detailed protocol to clearly and precisely identify and isolate both prominent and less-studied mouse adipose depots (Figure 1). This resource will facilitate a more complete investigation of the developmental, molecular, and functional characteristics of adipocytes within diverse niches.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59499/59499fig1.jpg" /> 
Figure 1: Schematic depiction of mouse adipose depots dissected in this protocol. This image has been adapted from Bagchi et al., 20184. <a href="https://www.jove.com/files/ftp_upload/59499/59499fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>10% neutral buffered formalin</td>
			<td>Fisher Scientific</td>
			<td>22-110-869</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates, untreated</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3738</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol (dilute from 95%)</td>
			<td>Fisher Scientific</td>
			<td>04-355-226</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting forceps with curved tips</td>
			<td>VWR</td>
			<td>89259-946</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting pan</td>
			<td>Carolina Biological Supply Company</td>
			<td>629004</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors (sharp/blunt tip)</td>
			<td>VWR</td>
			<td>82027-588</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze sponges</td>
			<td>Vitality Medical</td>
			<td>2634</td>
			<td>Curity 4 x 4 inch gauze sponge, 12 ply</td>
		</tr>
		<tr>
			<td>Handi-Pins for dissection</td>
			<td>Carolina Biological Supply Company</td>
			<td>629132</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris scissors (straight)</td>
			<td>VWR</td>
			<td>470018-890</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>VetOne</td>
			<td>501017</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>VWR</td>
			<td>100499-578</td>
			<td>Feather scalpel handle with blade, disposable</td>
		</tr>
	</tbody>
</table>

identification and dissection of diverse mouse adipose depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Successful identification and isolation of various mouse adipose depots can be achieved using the protocol described above. The gross anatomical locations of subcutaneous (A, E-F), brown (B), visceral (C, D, G-J), and popliteal (K) depots are shown in Figure 2.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59499/59499fig2.jpg" /> 
Figure 2: Gross anatomical locations of mou...

Watch this Scientific Journal Video about Identification and Dissection of Diverse Mouse Adipose Depots at JoVE.com

Identification and Dissection of Diverse Mouse Adipose Depots

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...

A standardized methodology for identifying and dissecting adipose depots throughout the body is critical for the investigation of differences in the character and function of adipocytes within discrete anatomical niches. The main advantage of this technique is that it is a reliable and accurate method for identifying and excising both common and less studied rodent adipose depots. For the identification and isolation of interscapular BAT, use iris scissors to cut horizontally along the bottom edge of the anterior subcutaneous tissue of a euthanized mouse following the natural border of the depot.Make two vertical incisions along the lateral edges of the depot following the natural borders of the tissue. Use forceps to carefully flip up the depot to reveal the butterfly-shaped interscapular brown adipose tissue embedded within the white adipose tissue. Then carefully dissect the BAT away from the surrounding WAT.Store adipose tissues in appropriate containers for downstream analysis. For the identification and isolation of posterior subcutaneous WAT, place the mouse into the supine position and secure the animal's limbs. Use forceps to lift up the skin at the base of the sternum and make a one millimeter skin incision.Insert one blade of the iris scissors into the cut and make a four to five centimeter midline skin incision beginning at the base of the sternum and ascending to the base of the tail. Next, make two one centimeter horizontal incisions at the top of the initial vertical incision extending laterally from the midline. Use the forceps to carefully peel back the skin from the peritoneal cavity, use the leg to locate the posterior subcutaneous WAT which should remain associated with the skin.Pin the outstretched skin and completely excise the triangular inguinal WAT depot from the base of the hindlimb ventrally across the groin. For visceral WAT depot identification and isolation, use forceps to lift up the thin peritoneal cavity wall at the base of the sternum and make a one millimeter incision in the tented tissue. Insert one blade of the iris scissors into the cut and make a four to five centimeter descending vertical incision from the top of the peritoneal cavity to the rectum.Make two one centimeter horizontal incisions at the top and bottom of the vertical incision extending laterally from the midline and use the forceps to peel back the peritoneum to expose the abdominal cavity contents. Then pin the outstretched peritoneum to the dissection pan to harvest the WAT depots contained within the peritoneal cavity. For the identification and isolation of visceral gonadal WAT, locate the gonads and use forceps to lift up the associated gonadal WAT, then use iris scissors to carefully excise the WAT from the gonads.To excise the perirenal depot, locate the kidney and use forceps to lift up the renal tissue. Pull the kidney to the midline to see a clear division between the perirenal and retroperitoneal depots and excise the WAT directly associated with the kidneys. Then remove the adrenal glands located above the kidneys from the WAT.For retroperitoneal WAT identification and isolation, lift the kidney to the midline again and use iris scissors to carefully dissect the retroperitoneal WAT from the posterior peritoneal wall. To isolate the popliteal WAT, place the patella against the dissection pan taking care that the popliteal fossa at the back of the knee is facing upward. Use the iris scissors to carefully remove the skin from the base of the hindlimb to the foot.Use the iris scissors to make a cut at the inferior border of the medial and lateral heads of the gastrocnemius muscle, use the forceps to lift up the muscle to reveal the triangular popliteal depot. Then use iris scissors to excise the depot along the natural tissue borders. Here, the gross anatomical locations of mouse subcutaneous, brown, visceral, and popliteal depots are shown.The histological characteristics of the subcutaneous, brown, visceral, popliteal, constitutive, and regulated marrow adipose, intramuscular, and infrapatellar adipose depots can be evaluated by standard Hematoxylin and Eosin staining protocols. The adipose tissues isolated using this protocol can be used for a wide variety of experimental endpoints including histological analysis, molecular studies, and gene and protein expression analysis. Field-wide standardization of the identification of diverse adipose depots will undoubtedly help us further our understanding of their molecular and metabolic characteristics and their differential contributions to both local and systematic pathological states.

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