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 mouse from the vaporizer chamber and confirm euthanasia using an approved secondary measure. 
	NOTE: Approved secondary measures will vary by institution and animal protocol and may include cervical dislocation or decapitation.</li>
	<li>Place the euthanized mouse on the dissection pan. Sterilize the dorsal and ventral external surfaces of the mouse using 70% ethanol. Ensure that the external surface of the mouse is sufficiently wet to minimize contamination from fur during dissection.</li>
</ol>
2. Identification and Isolation of Major Subcutaneous Adipose Ddepots (Anterior Subcutaneous, Dorsolumbar, Inguinal, Gluteal) and Interscapular Brown Aadipose Tissue
<ol>
	<li>Identifying and isolating anterior subcutaneous WAT 
	NOTE: Anterior subcutaneous WAT is located between the scapulae, descending from the nape of the neck to the axillae of the mouse7. This depot has alternatively been described as suprascapular8 or interscapular20 WAT and lies directly on top of the interscapular BAT depot.
	<ol>
		<li>To isolate the anterior subcutaneous depot, lay the mouse on its stomach in a prone position. Secure the upper and lower limbs to the dissection pan with dissection pins.</li>
		<li>Use forceps to lift the dorsal skin at the nape of the neck. Use iris scissors to make a small (1 mm) cut in the skin.</li>
		<li>Insert one blade of the iris scissors into the initial cut and make a midline vertical incision (2&#8211;3 cm) through the skin, beginning at the nape of the neck and descending along the spine to mid-back.</li>
		<li>Make two horizontal incisions (1 cm each) using the iris scissors, extending laterally from midline, at the top and bottom of the initial vertical incision.</li>
		<li>Use forceps to carefully peel back the skin and expose the anterior subcutaneous depot.</li>
		<li>Use iris scissors to remove the depot following the natural borders of the tissue. 
		NOTE: This method isolates both anterior subcutaneous WAT and interscapular BAT.</li>
		<li>Place the dissected depot on the dissection pan and carefully remove the contaminating BAT using iris scissors.</li>
	</ol>
	</li>
	<li>Identifying and isolating classical BAT 
	NOTE: Classical BAT is located beneath the anterior subcutaneous WAT depot.
	<ol>
		<li>To isolate BAT, use iris scissors to cut horizontally along the bottom edge of the anterior subcutaneous tissue, following the natural border of the depot.</li>
		<li>Then, use iris scissors to make two vertical incisions along the lateral edges of the depot, following the natural borders of the tissue.</li>
		<li>Use forceps to carefully flip the depot up and reveal the butterfly-shaped interscapular BAT embedded within the WAT. Carefully dissect the BAT out from the surrounding WAT.</li>
	</ol>
	</li>
	<li>Identifying and isolating posterior subcutaneous WAT,
	<ol>
		<li>Lay the mouse on its back in a supine position.</li>
		<li>After securing the upper and lower limbs with dissection pins, use forceps to lift up the skin at the base of the sternum and make a small (1 mm) cut in the skin.</li>
		<li>Insert one blade of the iris scissors into the initial cut and make a midline incision (4&#173;&#8211;5 cm) through the skin, beginning at the base of the sternum and descending to the base of the tail. Exercise caution when making this incision because the ventral skin is very thin and is closely associated with the underlying peritoneal cavity wall.</li>
		<li>Make two horizontal incisions (1 cm each) using iris scissors, extending laterally from midline, at the top of the initial vertical incision.</li>
		<li>Use forceps to carefully peel back the skin from the peritoneal cavity and the leg to find posterior subcutaneous WAT, which should remain associated with the skin. Secure the outstretched skin with dissection pins to ease complete excision of the WAT. 
		NOTE: Although the posterior subcutaneous WAT appears to be continuous, it is actually comprised of three discrete depots: dorsolumbar, inguinal, and gluteal1. The dorsolumbar depot extends from the lumbar spine to the base of the hindlimb. The triangular inguinal depot extends from the base of the hindlimb ventrally across the groin and contains a prominent lymph node. The gluteal depot extends inferiorly from the base of the groin and wraps around the leg to the base of the tail.</li>
	</ol>
	</li>
</ol>
3. Identification and Isolation of Visceral Adipose Depots (Gonadal, Perirenal, Retroperitoneal, Omental, Pericardial)
<ol>
	<li>Identifying and isolating visceral WAT depots 
	NOTE: WAT depots, which are largely contained within the thoracic and peritoneal cavities, keep the mouse in a supine position. Secure the upper and lower limbs to the dissection pan with dissection pins.
	<ol>
		<li>Use forceps to lift up the thin peritoneal cavity wall at the base of the sternum and make a small cut (1 mm) using iris scissors.</li>
		<li>Insert one blade of the iris scissors into the cut and make a descending vertical incision (4&#8211;5 cm) from the top of the peritoneal cavity (base of the sternum) to the rectum.</li>
		<li>Make two horizontal incisions (1 cm each) with iris scissors, extending laterally from midline, at the top and bottom of the vertical incision.</li>
		<li>Use forceps to peel back the peritoneum and expose the abdominal cavity contents. Pin the outstretched peritoneum to the dissection pan.</li>
	</ol>
	</li>
	<li>Identifying and isolating gonadal WAT 
	NOTE: Gonadal WAT surrounds the uterus and ovaries in females (ovarian or parametrial) and the epididymis and testes in males (epididymal). Ovarian WAT surrounds the ovaries, uterus, and bladder. In obese animals, gonadal and perirenal WAT can appear to be continuous &#8212; in this case, separate the depots at the uterine horn and ovaries. Epididymal WAT is found bound to the epididymis, testes, and the prominent epididymal blood vessel.
	<ol>
		<li>Locate the gonads (testes or ovaries) and use forceps to lift up the associated gonadal WAT.</li>
		<li>Use iris scissors to carefully excise the WAT from the gonads.</li>
	</ol>
	</li>
	<li>Identifying and isolating perirenal WAT 
	NOTE: Perirenal WAT surrounds the kidneys bilaterally. In obese animals, this depot can appear to extend inferiorly to the top of the uterine horn and ovaries. Although the perirenal WAT has traditionally been classified as a visceral depot21, several groups have identified it as a brown depot based on lineage tracing and radiolabeled glucose uptake studies8,9,20. Histologically it is comprised of a mixture of white and brown adipocytes.
	<ol>
		<li>To excise the perirenal depot, locate the kidney and use forceps to lift it up and pull it midline to see a clear division between the perirenal and retroperitoneal depots.</li>
		<li>Excise the WAT directly associated with the kidneys. Ensure that the adrenal glands, located above the kidneys, are removed from the WAT.</li>
	</ol>
	</li>
	<li>Identifying and isolating retroperitoneal WAT 
	NOTE: Retroperitoneal WAT is located in a paravertebral position, along the border between the posterior abdominal wall and the spinal cord.
	<ol>
		<li>To excise this depot, use forceps to lift the kidney up and towards midline to clearly see the border between the perirenal and retroperitoneal depots. 
		NOTE: In obese animals, identifying this border can be challenging.</li>
		<li>Then, use iris scissors to carefully dissect the retroperitoneal WAT from the posterior peritoneal wall.</li>
	</ol>
	</li>
	<li>Identifying and isolating omental WAT 
	NOTE: Omental WAT is located along the greater curvature of the stomach. Although omental adipose is an important depot in humans, it is generally present only in morbidly obese mice.
	<ol>
		<li>To identify visceral omental WAT, use forceps to lift the stomach up. Use iris scissors to remove the associated adipose tissue along the inferior border of the stomach. Do not confuse omental WAT with the pancreas.</li>
	</ol>
	</li>
	<li>Identifying and isolating mesenteric WAT 
	NOTE: Mesenteric WAT is a web-like structure surrounding and associated with the small and large intestines.
	<ol>
		<li>To excise this depot, remove the intestines from the rest of the digestive tract by cutting at the base of the stomach and the rectum. Use forceps to lift the intestines out of the visceral cavity and unravel them.</li>
		<li>Use iris scissors to carefully dissect the mesenteric WAT away from the intestines, beginning at the duodenum and continuing to the end of the colon. Carefully remove lymph nodes that are closely associated with the mesenteric depot.</li>
	</ol>
	</li>
	<li>Identifying and isolating pericardial WAT 
	NOTE: Pericardial WAT is located outside the visceral pericardium and on the external surface of the parietal pericardium, often along the inferior aspect of the heart14.
	<ol>
		<li>To gain access to the thoracic cavity, keep the mouse in a supine position. Secure the upper and lower limbs to the dissection pan with dissection pins.</li>
		<li>Use forceps to lift the xyphoid process (cartilage at the base of the sternum) and make a small (1 mm) cut in the thoracic cavity wall.</li>
		<li>Insert one blade of the iris scissors into the cut and make two horizontal incisions (1 cm each) extending laterally from the base of the sternum. This will expose the diaphragm and inferior border of the thoracic cavity.</li>
		<li>Use dissecting scissors to make two ascending vertical incisions (3&#8211;4 cm) through the ribcage, extending superiorly from the lateral borders of the thoracic cavity to the clavicle.</li>
		<li>Use forceps to lift up the ventral half of the ribcage. Use dissecting scissors to make a final cut (2 cm) along the inferior length of the clavicle to remove the ribcage and expose the thoracic cavity contents.</li>
		<li>If present, carefully dissect the pericardial adipose from the outer surface of the pericardium.</li>
	</ol>
	</li>
</ol>
4. Identification and Isolation of Other Adipose Depots
<ol>
	<li>Identifying and isolating epicardial WAT 
	NOTE: Epicardial WAT is contained within the visceral pericardium and is directly associated with the surface of the myocardium.
	<ol>
		<li>If present, epicardial adipocytes can be observed histologically after isolation and fixation of the perfused heart in 10% neutral buffered formalin for 24 h.</li>
	</ol>
	</li>
	<li>Identifying and isolating popliteal WAT 
	NOTE: Popliteal WAT is located in the popliteal fossa in the posterior knee and contains a large lymph node. This depot is not typically visible in young animals.
	<ol>
		<li>To isolate popliteal WAT, use iris scissors to carefully remove the skin from the base of the hind limb to the foot.</li>
		<li>Place the patella against the dissection pan and secure the outstretched leg with a pin in the foot. Ensure that the popliteal fossa at the back of the knee is facing upward.</li>
		<li>Use iris scissors to make a cut at the inferior border of the medial and lateral heads of the gastrocnemius muscle. Use forceps to lift up the muscle and reveal the triangular popliteal depot.</li>
		<li>Use iris scissors to excise the depot along the natural tissue borders.</li>
	</ol>
	</li>
	<li>Identifying and isolating dermal WAT 
	NOTE: Dermal WAT is a thin layer of adipocytes located between the reticular dermis and the panniculus carnosus muscle layer.
	<ol>
		<li>To identify dermal adipocytes by histological methods, place the mouse on its stomach in a prone position. After securing the upper and lower limbs with dissection pins, spray the external surface of the mouse with 70% ethanol to wet the skin.</li>
		<li>Use a scalpel to remove the wetted fur from a square portion of skin on the back of the mouse.</li>
		<li>Use iris scissors to carefully excise the shaved portion of skin, which will include the reticular dermis, dermal WAT, panniculus carnosus, and some subcutaneous WAT.</li>
		<li>Use a scalpel to cut the excised skin into thin vertical strips.</li>
		<li>Position the thin vertical strips with the sticky subcutaneous WAT layer facing down.</li>
		<li>Starting at one end, roll the strip onto itself to form a spiral. The sticky subcutaneous WAT layer on the outside of the spiral will allow the roll to maintain its shape during fixation.</li>
		<li>Place the spiral in a well of a 24 well plate containing 10% neutral buffered formalin for 24 h prior to histological processing22.</li>
	</ol>
	</li>
	<li>Identifying and isolating intermuscular WAT 
	NOTE: Intermuscular WAT is broadly defined as adipocytes located beneath the deep fascia of muscles. This term includes adipocytes interspersed between muscle fibers of skeletal muscle, also known as intramuscular WAT, and adipocytes located within muscle bundles themselves. Wildtype mice generally do not have large numbers of intermuscular adipocytes. However, it is possible under certain conditions to identify intermuscular WAT by histological methods. Under some conditions, intermuscular adipocytes can also be found in smooth muscles, such as the diaphragm.
	<ol>
		<li>To isolate the tibialis anterior (TA) muscle, for example, place the mouse on its back in a supine position and secure the lower limbs to the dissection pan using dissection pins. Wet the skin with 70% ethanol to minimize contamination with fur.</li>
		<li>Use iris scissors to carefully remove the skin from the leg and expose the quadriceps muscle group, located above the knee on the femur shaft, and the TA located below the knee on the ventral surface of the tibia. 
		NOTE: The TA is thick near the proximal end of the tibia and more tendinous near the distal end.</li>
		<li>Use iris scissors to excise a piece of the muscle and fix in 10% neutral buffered formalin for 24 h prior to histological processing22.</li>
	</ol>
	</li>
	<li>Identifying and isolating intra-articular WAT depots 
	NOTE: Intra-articular WAT depots are located within synovial joints.
	<ol>
		<li>To identify infrapatellar WAT, for example, by histological methods, harvest leg bones as detailed in the BMAT section.</li>
		<li>After the removing the femur-tibial complex, use iris scissors and gauze pads to remove as much muscle and connective tissue as possible. Do not break the tibiofemoral joint.</li>
		<li>Fix the femur-tibial complex in 10% neutral buffered formalin for 24 h prior to decalcification and histological processing (described below, step 4.6.8).</li>
	</ol>
	</li>
	<li>Identifying and isolating bone marrow adipose tissue 
	NOTE: Bone marrow adipose tissue (BMAT) is contained within bones, interspersed with hematopoietic cells. Anatomically, BMAT can be classified as constitutive (distal tibia and caudal vertebrae) or regulated (mid-to-proximal tibia, femur, and lumbar vertebrae)10,11. Clean isolation of bone marrow adipocytes for RNA and protein analyses is challenging in mice. However, mouse bones can readily be harvested, fixed, decalcified and paraffin-embedded for histological analyses.
	<ol>
		<li>To harvest leg bones, for example, first remove the legs from the mouse. Use dissection scissors to cut the acetabulofemoral joint, keeping the femoral head intact.</li>
		<li>Use iris scissors to carefully remove the skin from the leg, revealing the leg muscles.</li>
		<li>Carefully dissect away the main muscles from the femur and tibia using iris scissors and gauze pads.</li>
		<li>When the femur is exposed, follow the edge of the bone to the articulation of the pelvis and femur and carefully release the femoral head from the acetabulofemoral joint. Use gauze to clean any remaining tissue from the femur.</li>
		<li>Follow the border of the tibia to the ankle joint. Carefully release the medial malleolus, located at the tip of the tibia, from the ankle joint.</li>
		<li>Once the femur-tibial complex has been isolated, remove as much muscle and connective tissue as possible using iris scissors and/or gauze.</li>
		<li>Separate the tibia from the femur by inserting one blade of the iris scissors into the tibiofemoral joint and gently cut through the medial and lateral collateral ligaments and the anterior and posterior cruciate ligaments. Do not remove the capsule of the knee joint to ensure that the tibia remains intact. Use gauze to clean any remaining tissue from the tibia.</li>
		<li>Fix bones in 10% neutral buffered formalin for 24 h at room temperature and then wash and store according to future needs.
		<ol>
			<li>To analyze bone parameters using microcomputed tomography (&#181;CT), store bones in Sorensen&#8217;s phosphate buffer, pH 7.423.</li>
			<li>For BMAT quantification and histological analyses, decalcify bones in 14% EDTA, pH 7.4, for 10-14 days.</li>
			<li>Following decalcification, use osmium tetroxide staining and &#181;CT analysis to quantify BMAT24. Otherwise, process and paraffin-embed bones for histology23.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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<li>Scheller, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Region-specific+variation+in+the+properties+of+skeletal+adipocytes+reveals+regulated+and+constitutive+marrow+adipose+tissues%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Use+of+osmium+tetroxide+staining+with+microcomputerized+tomography+to+visualize+and+quantify+bone+marrow+adipose+tissue+in+vivo%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">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/pubmed?term=((Genomic+profiling+reveals+that+transient+adipogenic+activation+is+a+hallmark+of+mouse+models+of+skeletal+muscle+regeneration%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Muscle+Regeneration+with+Intermuscular+Adipose+Tissue+%28IMAT%29+Accumulation+Is+Modulated+by+Mechanical+Constraints%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Intermuscular+and+perimuscular+fat+expansion+in+obesity+correlates+with+skeletal+muscle+T+cell+and+macrophage+infiltration+and+insulin+resistance%5BTitle%5D)+AND+%22International+Journal+of+Obesity%22%5BJournal%5D)">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. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+Circulating+Adiponectin+in+Response+to+Thiazolidinediones%3A+Investigating+the+Role+of+Bone+Marrow+Adipose+Tissue%5BTitle%5D)+AND+%22Frontiers+in+Endocrinology%22%5BJournal%5D)">Increased Circulating Adiponectin in Response to Thiazolidinediones: Investigating the Role of Bone Marrow Adipose Tissue.</a> Frontiers in Endocrinology. 7, 128(2016).</li>
</ol>

The authors have nothing to disclose.

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 investigators18,19. This manuscript is a novel and important contribution to the field of adipose biology since it provides an in-depth guide to the anatomic location and precise dissection of commonly studied and lesser known depots throughout the mouse. Gross anatomical location and histological analyses are provided to demonstrate the diversity of adipocyte niches.
Successful isolation of discrete adipose depots using this protocol is dependent on several critical steps. Maintenance of a clean dissection environment is crucial; 70% ethanol can be used as necessary to remove contaminating hairs or blood from the dissection tray and tools. When isolating posterior subcutaneous WAT, it is imperative that the initial incision in the skin does not penetrate the closely associated peritoneal cavity so that the two layers can be efficiently separated to expose the WAT. Similarly, when cutting through the peritoneal wall to expose the abdominal cavity contents, incisions must not perforate the underlying intestines to prevent contamination with digestive elements. Interscapular BAT must be carefully excised from the surrounding tissue to prevent WAT contamination, which will skew future analyses. Consistent identification of the posterior subcutaneous dorsolumbar, inguinal, and gluteal depots depends on the utilization of precise anatomic landmarks described in the protocol. The distinction between these depots is particularly important; although the depots can appear to be continuous, centrally located inguinal adipocytes acquire brown-like characteristics more readily than their surrounding counterparts.
The adipose tissues isolated using this protocol can be analyzed using a variety of techniques. In addition to histological evaluation (e.g. H&#38;E staining, immunohistochemistry), adipose tissues can be used for a wide array of molecular analyses, from regulation of transcription through to posttranslational protein modifications. Adipocytes and stromal vascular cells within individual depots can be isolated by collagenase digestion and differential centrifugation. These fractions can then be used for molecular, metabolic, and&#160;cell culture studies. Depot explants can also be used for ex vivo metabolic and enzymatic assays.
Several challenges and limitations arise during the isolation and excision of mouse adipose tissues. First, some depots that are physiologically important in humans are not commonly present in lean, adult mice. For example, omental WAT, which is the major visceral depot in humans, can only be seen in genetically or diet-induced obese mice. The expansion of some depots can, however, be induced by nutritional challenges or drug treatments. For example, high fat diet feeding can cause the expansion of omental and pericardial WAT. Intermuscular WAT can be induced by skeletal muscle injury25,26 or high fat diet27. Regulated BMAT expands in response to various challenges, including high fat diet, calorie restriction and thiazolidinedione treatment11,28. Second, due to the small size of mice, obtaining enough sample for molecular or metabolic analyses can be difficult. For example, isolation of enough pure bone marrow adipocytes for subsequent analyses is challenging, even after pooling bones from multiple mice. Third, accurately defining borders of certain depots can be difficult. For example, posterior subcutaneous WAT appears to be one continuous depot but is actually comprised of discrete dorsolumbar, inguinal, and gluteal depots. Additionally, the perirenal can appear to be fused to both the gonadal and retroperitoneal depots in very obese animals. The lack of distinct borders between adjacent depots can make clean isolation of these tissues challenging. However, this dissection protocol provides investigators with a detailed atlas and step-by-step guidance for accurate and reproducible dissection of a wide range of mouse adipose depots. Field-wide standardization of the identification and isolation of discrete mouse adipose depots described above will undoubtedly help further elucidate differences in the development, gene expression, and local and systemic functions of previously under-studied adipocyte niches.

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 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="Rodent fat tissue distribution, anatomical diagram, showing subcutaneous and visceral fat depots." class="xfigimg" src="/files/ftp_upload/59499/59499fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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.remotexs.ntu.edu.sg/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 inch 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="Rodent dissection process; anatomical structures labeled in diagrams for educational study." class="xfigimg" src="/files/ftp_upload/59499/59499fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Gross anatomical locations of mouse adipose depots. Gross anatomy of (A) anterior subcutaneous, (B) brown, (C) epididymal (b, bladder), (D) ovarian (u, uterine horns), (E) posterior subcutaneous (dorsolumbar (d), inguinal (i), gluteal (g)), (F) inguinal, (G) mesenteric, (H) perirenal (k, kidney), (I) retroperitoneal (k, kidney), (J) pericardial (h, heart), and (K) popliteal adipose depots in C57BL/6J adult mice. Arrows point to specific depots if multiple depots are depicted. Relevant organs are designated by appropriate letters. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59499/59499fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Histological characteristics of subcutaneous (A-D), brown (E), visceral (F-K), popliteal (L), constitutive (M) and regulated marrow adipose (N), intramuscular (O), and infrapatellar (P) adipose depots were evaluated by Hematoxylin and Eosin (H&amp;E) staining22 and are shown in Figure 3.
<img alt="Histology results showing various tissue samples with adipose and connective tissue structures." class="xfigimg" src="/files/ftp_upload/59499/59499fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Histological evaluation of discrete mouse adipose depots. Histology of (A) anterior subcutaneous, (B) dorsolumbar, (C) inguinal, (D) gluteal, (E) brown, (F) gonadal, (G) perirenal, (H) retroperitoneal, (I) omental, (J) mesenteric, (K) pericardial, (L) popliteal, (M) constitutive and (N) regulated bone marrow adipose, (O) intermuscular, and (P) infrapatellar depots in C57BL/6J adult mice. Tissues were isolated according to the given protocol, fixed overnight in 10% neutral buffered formalin, processed, and embedded in paraffin. 5 µm sections were stained with H&amp;E. Most images were taken at 100x magnification; Scale bar = 100 µm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59499/59499fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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, ...

identification-and-dissection-of-diverse-mouse-adipose-depots

Nanyang Technological University

Research

JoVE Journal

Biology

40.3K Views.  University of Michigan Medical School. Een gestandaardiseerde methodologie voor het identificeren en ontleden van vetdepots door het hele lichaam is van cruciaal belang voor het onderzoek naar verschillen in het karakter en de functie van adipocyten binnen discrete anatomische niches. Het belangrijkste voordeel van deze techniek is dat het een betrouwbare en nauwkeurige methode is voor het identificeren en uitsnijden van zowel gemeenschappelijke als minder bestudeerde vetdepots van knaagdieren. Voor de identificatie en isolatie va...

Video: Identification and Dissection of Diverse Mouse Adipose Depots

Mouse Adipose Tissue Collection and Processing for RNA Analysis

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and Western Blot, since it mostly contains lipids. RNA isolation from adipose tissue samples is also challenging as extra steps are required to avoid these lipids. Here, we present a procedure to collect three anatomically different white adipose tissues from mice, to process these samples and perform RNA isolation. We further describe the synthesis of cDNA and gene expression experiments using real-time PCR. The hereby described protocol allows the reduction of contamination from the animal's hair and blood on fat pads as well as cross-contamination between different fat pads during tissue collection. It has also been optimized to ensure adequate quantity and quality of the RNA extracted. This protocol can be widely applied to any mouse model where adipose tissue samples are required for routine experiments such as real-time PCR but is not intended for isolation from primary adipocytes cell culture.

The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA.

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

Biochemistry

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

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Immunology and Infection

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 ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

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 ...

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...

Morphological and Molecular Characterization of the scBAT in Mice

Dissection, Histological Processing, and Gene Expression Analysis of Murine Supraclavicular Brown Adipose Tissue

Brown adipose tissue (BAT)-mediated thermogenesis plays an important role in the regulation of metabolism, and its morphology and function can be greatly impacted by environmental stimuli in mice and humans. Currently, murine interscapular BAT (iBAT), which is located between two scapulae in the upper dorsal flank of mice, is the main BAT depot used by research laboratories to study BAT function. Recently, a few previously unknown BAT depots were identified in mice, including one analogous to human supraclavicular brown adipose tissue. Unlike iBAT, murine supraclavicular brown adipose tissue (scBAT) is situated in the intermediate layer of the neck and thus cannot be accessed as readily.
To facilitate the study of newly identified mouse scBAT, presented herein is a protocol detailing the steps to dissect intact scBAT from postnatal and adult mice. Due to scBAT&#39;s small size relative to other adipose depots, procedures have been modified and optimized specifically for processing scBAT. Among these modifications is the use of a dissecting microscope during tissue collection to increase the precision and homogenization of frozen scBAT samples to raise the efficiency of subsequent qPCR analysis. With these optimizations, the identification of, morphological appearance of, and molecular characterization of the scBAT can be determined in mice.

Author Spotlight: Optimizing Supraclavicular Brown Adipose Tissue Extraction for Genetic Analysis

Here, we provide a practical procedure for dissecting and performing histological and gene expression analyses of murine supraclavicular brown adipose tissue.

Brown adipose tissue (BAT)-mediated thermogenesis plays an important role in the regulation of metabolism, and its morphology and function can be greatly ...

Culturing Murine Adipocytes and Explants for Functional and Imaging Studies

Isolation and Culturing of Primary Murine Adipocytes from Lean and Obese Mice

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and mobilization, thermoregulation, and the secretion of endocrine factors. The expansion of white adipose tissue due to caloric imbalance results in both the enlargement of existing adipocytes and the generation of additional adipocytes from adipocyte progenitor cells. Obesity-driven changes to white adipose tissue, including those affecting adipocytes, are associated with numerous comorbidities, such as type 2 diabetes and 13 types of cancer. A significant barrier to studying how adipocytes contribute to disease is the inability to readily isolate and culture mature adipocytes. This article describes a protocol to isolate murine lean and obese adipocytes from the subcutaneous and visceral fat depots of male and female C57BL/6 mice. The protocol details how isolated primary adipocytes can be cultured in a membrane adipocyte aggregate system for up to 2 weeks, facilitating their functional analysis in co-culture experiments, lipolysis assays, or through the collection of conditioned media containing adipocyte-secreted factors. Additionally, the protocol outlines methods for culturing adipose tissue explants in basement membrane matrix domes and imaging primary isolated adipocytes. Importantly, this approach can be integrated with existing protocols for the isolation of adipose tissue-resident adipocyte&#160;progenitor cells using fluorescence-activated cell sorting (FACS). Together, these protocols provide researchers with tools to functionally study adipocytes, adipocyte progenitor cells, and whole adipose tissue from lean and obese, male and female mice.

The size and fragility of mature adipocytes have limited the techniques and tools available for their study and isolation. Here, a protocol is described for the isolation of mature murine adipocytes, which can be easily adapted for different adipose depots and mouse diets.

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and ...

Identification and Dissection of Diverse Mouse Adipose Depots

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