This work is supported by NIDDK of the NIH under Award Number R01DK116899, USDA/ARS under Award Number 3092-51000-064-000D, and a pilot award from the Baylor College of Medicine Cardiovascular Research Institute. The flowcharts were produced using BioRender.

The animal procedures were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine. All procedures were performed on C57BL/6J male mice aged 3 weeks and 3 months old. Prior to the dissection, all mice were euthanized using the approved rodent carbon dioxide euthanasia procedure. See the Table of Materials for details related to all materials, reagents, and instruments used in this protocol.
1. Dissection of scBAT
<ol>
	<li>Place the mouse ca...

Morphological and Molecular Characterization of the scBAT in Mice

<ol>
	<li>Boutari, C., Mantzoros, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+2022+update+on+the+epidemiology+of+obesity+and+a+call+to+action:+as+its+twin+COVID-19+pandemic+appears+to+be+receding,+the+obesity+and+dysmetabolism+pandemic+continues+to+rage+on.">A 2022 update on the epidemiology of obesity and a call to action: as its twin COVID-19 pandemic appears to be receding, the obesity and dysmetabolism pandemic continues to rage on.</a> Metabolism. 133, 155217 (2022).</li><li>Hales, C. M., Carroll, M. D., Fryar, C. D., Ogden, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+obesity+and+severe+obesity+among+adults:+United+States,+2017-2018.">Prevalence of obesity and severe obesity among adults: United States, 2017-2018.</a> NCHS Data Brief. (360), 1-8 (2020).</li><li>Berry, D. C., Stenesen, D., Zeve, D., Graff, J. 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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=Identification+and+importance+of+brown+adipose+tissue+in+adult+humans.">Identification and importance of brown adipose tissue in adult humans.</a> N Engl J Med. 360 (15), 1509-1517 (2009).</li><li>van Marken Lichtenbelt, W. D., 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=Cold-activated+brown+adipose+tissue+in+healthy+men.">Cold-activated brown adipose tissue in healthy men.</a> N Engl J Med. 360 (15), 1500-1508 (2009).</li><li>Virtanen, K. A., 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=Functional+brown+adipose+tissue+in+healthy+adults.">Functional brown adipose tissue in healthy adults.</a> N Engl J Med. 360 (15), 1518-1525 (2009).</li><li>Cypess, A. 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=Anatomical+localization,+gene+expression+profiling+and+functional+characterization+of+adult+human+neck+brown+fat.">Anatomical localization, gene expression profiling and functional characterization of adult human neck brown fat.</a> Nat Med. 19 (5), 635-639 (2013).</li><li>Leitner, B. P., 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+human+brown+adipose+tissue+in+lean+and+obese+young+men.">Mapping of human brown adipose tissue in lean and obese young men.</a> Proc Natl Acad Sci U S A. 114 (32), 8649-8654 (2017).</li><li>Mo, Q., 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=Identification+and+characterization+of+a+supraclavicular+brown+adipose+tissue+in+mice.">Identification and characterization of a supraclavicular brown adipose tissue in mice.</a> JCI Insight. 2 (11), e93166 (2017).</li><li>Shi, 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=Gene+Expression+Analysis+of+Environmental+Temperature+and+High-Fat+Diet-Induced+Changes+in+Mouse+Supraclavicular+Brown+Adipose+Tissue.">Gene Expression Analysis of Environmental Temperature and High-Fat Diet-Induced Changes in Mouse Supraclavicular Brown Adipose Tissue.</a> Cells. 10 (6), 1370 (2021).</li><li>Chang, 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=Loss+of+perivascular+adipose+tissue+on+peroxisome+proliferator-activated+receptor-gamma+deletion+in+smooth+muscle+cells+impairs+intravascular+thermoregulation+and+enhances+atherosclerosis.">Loss of perivascular adipose tissue on peroxisome proliferator-activated receptor-gamma deletion in smooth muscle cells impairs intravascular thermoregulation and enhances atherosclerosis.</a> Circulation. 126 (9), 1067-1078 (2012).</li><li>Ye, 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=Developmental+and+functional+characteristics+of+the+thoracic+aorta+perivascular+adipocyte.">Developmental and functional characteristics of the thoracic aorta perivascular adipocyte.</a> Cell Mol Life Sci. 76 (4), 777-789 (2019).</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> Am J Physiol Endocrinol Metab. 308 (12), E1085-E1105 (2015).</li><li>Fu, 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=Neural+crest+cells+differentiate+into+brown+adipocytes+and+contribute+to+periaortic+arch+adipose+tissue+formation.">Neural crest cells differentiate into brown adipocytes and contribute to periaortic arch adipose tissue formation.</a> Arterioscler Thromb Vasc Biol. 39 (8), 1629-1644 (2019).</li><li>Tucker, D. K., Foley, J. F., Bouknight, S. A., Fenton, S. 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In this protocol, we present in detail the procedures for dissecting and processing scBAT for H&#38;E and gene expression analyses. Because scBAT resides in the intermediate layer of the neck and lies along the large veins, the isolation of this depot requires precise technique. Specifically, to gain a clear view of the depot, we recommend placing the mouse under a dissecting microscope after the neck has been opened. Using a pair of superfine point forceps to peel scBAT off the salivary gland and surrounding veins, care...

The increasing prevalence of obesity in the U.S. and worldwide has ignited great interest in understanding its etiology and identifying potential treatments1,2. Adipose tissue plays a vital role in metabolism, and dysregulation of the adipose tissue can lead to the development of obesity. Generally, there are two types of adipose tissues, white and brown adipose tissue. While white adipose tissue (WAT) can store chemical energy and secrete endocrine factors, brown adipose tissue (BAT) can use chemical energy to generate heat and maintain body temperature in the cold3,4. Because of this unique ability, activation of BAT can also increase energy expenditure and improve insulin sensitivity5.
BAT exerts its function through non-shivering thermogenesis, a process mediated by uncoupling protein 1 (UCP1)6. Mammals, including mice and humans, possess varying amounts of BAT. The classical view of BAT is that these adipose tissues are more abundant in mice and infants than in adult humans. iBAT, located in the upper dorsal flank between the scapulae, is the most studied BAT depot in mice. By applying radioisotope imaging and biopsy tests, recent studies identified several BAT depots in adult humans. Some of them, including the depots found in the deep neck and supraclavicular region, had not previously been identified in mice or other model animals7,8,9,10,11. Among these BAT depots, the scBAT is the most frequently seen depot in adult humans. To better understand the origin and molecular contribution of these newly found BAT depots in humans, it is essential to identify equivalent depots in mice that allow genetic and molecular manipulations to trace and test the functional role of these depots. Thus, we and others identified a few previously unknown BAT depots in different anatomical locations in mice, including scBAT12,13, thoracic perivascular BAT14,15, perirenal BAT16, and periaortic BAT17. Mouse scBAT anatomically resembles human scBAT and morphologically resembles classical iBAT, expressing high levels of UCP112.
Unlike mouse iBAT, which can be readily dissected, scBAT is situated in the intermediate layer of the mouse neck, beneath the salivary glands and along the external jugular vein. Isolation of this depot for histological and molecular analyses can be challenging. Here, we describe in detail the procedure for dissecting scBAT from postnatal and adult mice and processing this depot for histology and gene expression analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>95% Dehydrant Alcohol (Flex 95)</td>
			<td>Epredia</td>
			<td>8201</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Dehydrant Alcohol (Flex 100)</td>
			<td>Epredia</td>
			<td>8101</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well PCR plate</td>
			<td>Bio-Rad</td>
			<td>MLL9601</td>
			<td></td>
		</tr>
		<tr>
			<td>Aurum Binding Mini Column</td>
			<td>Bio-Rad</td>
			<td>7326826</td>
			<td></td>
		</tr>
		<tr>
			<td>Aurum High Stringency Wash</td>
			<td>Bio-Rad</td>
			<td>7326803</td>
			<td></td>
		</tr>
		<tr>
			<td>Aurum Low Stringency Wash</td>
			<td>Bio-Rad</td>
			<td>7326804</td>
			<td></td>
		</tr>
		<tr>
			<td>Base Molds (for embedding)</td>
			<td>Tissue-Tek</td>
			<td>4122</td>
			<td></td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle 21g x 1 1/2&#34;</td>
			<td>Becton Dickinson</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1840148</td>
			<td></td>
		</tr>
		<tr>
			<td>Capless Microcentrifuge Tubes 2 mL</td>
			<td>Fisherbrand</td>
			<td>02-681-453</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5430R</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX Opus 96 Real-Time PCR Instrument</td>
			<td>Bio-Rad</td>
			<td>12011319</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Thermo Scientific Chemicals</td>
			<td>383760010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytoseal 60 Low-viscosity mounting medium</td>
			<td>Epredia</td>
			<td>83104</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC-Treated Water</td>
			<td>Ambion</td>
			<td>AM 9906</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Nikon</td>
			<td>SMZ1500</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase Dilution Solution</td>
			<td>Bio-Rad</td>
			<td>7326805</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Bio-Rad</td>
			<td>7326828</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Invitrogen</td>
			<td>18427013</td>
			<td></td>
		</tr>
		<tr>
			<td>Elution solution</td>
			<td>Bio-Rad</td>
			<td>7326801</td>
			<td></td>
		</tr>
		<tr>
			<td>EM 400 embedding medium paraffin</td>
			<td>Leica Biosystems</td>
			<td>3801320</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y (0.5% w/v)</td>
			<td>RICCA</td>
			<td>2858-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Formula R Infiltration medium paraffin</td>
			<td>Leica Biosystems</td>
			<td>3801470</td>
			<td></td>
		</tr>
		<tr>
			<td>Genemark Nutator Gyromixer 349</td>
			<td>Bio Express</td>
			<td>S-3200-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Gill #3 Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>GHS332-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl (for HCL-Ethanol)</td>
			<td>Fisher Chemical</td>
			<td>A142212</td>
			<td></td>
		</tr>
		<tr>
			<td>IP VI Embedding Cassettes</td>
			<td>Leica Biosystems</td>
			<td>39LC-550-5-L</td>
			<td></td>
		</tr>
		<tr>
			<td>Koptec&#39;s Pure Ethanol - 200 Proof (for 70% Ethanol)</td>
			<td>Decon Labs</td>
			<td>V1001</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 (25 mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0971</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes 1.7 mL</td>
			<td>Avantor</td>
			<td>87003-294</td>
			<td></td>
		</tr>
		<tr>
			<td>Microseal &#39;B&#39; Seals (adhseive seals)</td>
			<td>Bio-Rad</td>
			<td>MSB1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems</td>
			<td>RM2245</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology Grade Water</td>
			<td>Corning</td>
			<td>46-000-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar Coors Tek</td>
			<td>Thomas Scientific</td>
			<td>60310</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl (for 0.85% saline)</td>
			<td>Fisher Bioreagents</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop Spectrophotometer</td>
			<td>NanoDrop Technologies</td>
			<td>ND-1000 UV/Vis</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo dT</td>
			<td>Invitrogen</td>
			<td>18418020</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Section Flotation Bath</td>
			<td>Boekel Scientific</td>
			<td>14792V</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>P6148-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Tube Strip</td>
			<td>Avantor</td>
			<td>76318-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle by Coors Tek</td>
			<td>Thomas Scientific</td>
			<td>60311</td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle Pellet Motor</td>
			<td>Kimble</td>
			<td>749540-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Model 19 Vacuum Oven&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>CAT# 51221162</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer: 36B4&#160; (forward) 10 &#956;M 
			5&#39; TGA AGT GCT CGA CAT CAC AGA GCA 3&#8217;</td>
			<td></td>
			<td></td>
			<td>Chen lab Oligo database</td>
		</tr>
		<tr>
			<td>Primer: 36B4 (reverse) 10 &#956;M 
			5&#39; GCT TGT ACC CAT TGA TGA TGG AGT GT 3&#8217;</td>
			<td></td>
			<td></td>
			<td>Chen lab Oligo database</td>
		</tr>
		<tr>
			<td>Primer: Fabp4 (forward) 10 &#956;M 
			5&#8217; ACA CCG AGA TTT CCT TCA AAC TG 3&#8217;</td>
			<td></td>
			<td></td>
			<td>Chen lab Oligo database</td>
		</tr>
		<tr>
			<td>Primer: Fabp4 (reverse) 10 &#956;M 
			5&#8217; CCA TCT AGG GTT ATG ATG CTC TTC A 3&#8217;</td>
			<td></td>
			<td></td>
			<td>Chen lab Oligo database</td>
		</tr>
		<tr>
			<td>Primer: Glut 4 (forward primer) 10 &#956;M 
			5&#8217; CTG ATT CTG CTG CCC TTC TGT CCT 3&#8217;</td>
			<td></td>
			<td></td>
			<td>Chen lab Oligo database</td>
		</tr>
		<tr>
			<td>Primer: Glut 4 (reverse) 10 &#956;M 
			5&#8217; GAC ATT GGA CGC TCT CTC TCC AAC TT 3&#8217;</td>
			<td></td>
			<td></td>
			<td>Chen lab Oligo database</td>
		</tr>
		<tr>
			<td>Primer: PPARg (forward) 10 &#956;M 
			5&#8217; AGG GCG ATC TTG ACA GGA AAG ACA 3&#8217;</td>
			<td></td>
			<td></td>
			<td>Chen lab Oligo database</td>
		</tr>
		<tr>
			<td>Primer: PPARg (reserve) 10 &#956;M 
			5&#8217; AAA TTC GGA TGG CCA CCT CTT TGC 3&#8217;</td>
			<td></td>
			<td></td>
			<td>Chen lab Oligo database</td>
		</tr>
		<tr>
			<td>Primer: Ppargc1a (reverse) 10 &#956;M 
			5&#39; ATG TTG CGA CTG CGG TTG TGT ATG 3&#8217;</td>
			<td></td>
			<td></td>
			<td>Chen lab Oligo database</td>
		</tr>
		<tr>
			<td>Primer: Ppargc1a(forward) 10 &#956;M 
			5&#39; ACG TCC CTG CTC AGA GCT TCT CA 3&#8217;</td>
			<td></td>
			<td></td>
			<td>Chen lab Oligo database</td>
		</tr>
		<tr>
			<td>Primer: Ucp1 (forward) 10 &#956;M 
			5&#8217; AGC CAC CAC AGA AAG CTT GTC AAC 3&#8217;</td>
			<td></td>
			<td></td>
			<td>Chen lab Oligo database</td>
		</tr>
		<tr>
			<td>Primer: Ucp1 (reverse) 10 &#956;M 
			5&#8217; ACA GCT TGG TAC GCT TGG GTA CTG 3&#8217;</td>
			<td></td>
			<td></td>
			<td>Chen lab Oligo database</td>
		</tr>
		<tr>
			<td>RNA isolation solution (PureZol)</td>
			<td>Bio-Rad</td>
			<td>7326880</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Away (surface decontaminant)</td>
			<td>Thermo Scientific</td>
			<td>1437535</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase H</td>
			<td>NEB</td>
			<td>M0297S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase inhibitor (RNase Out)</td>
			<td>Invitrogen</td>
			<td>10777019</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation Vial (glass)</td>
			<td>Electron Microscopy Sciences</td>
			<td>72632</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide drying bench&#160;</td>
			<td>Electrothermal (Cole-Parmer)</td>
			<td>MH6616</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless staining rack</td>
			<td>Electron Microscopy Sciences</td>
			<td>70312-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope (for embedding)</td>
			<td>Olympus</td>
			<td>SZ51</td>
			<td></td>
		</tr>
		<tr>
			<td>Sugical scissors</td>
			<td>McKesson</td>
			<td>43-1-104</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfine point Straight Dissecting Forceps</td>
			<td>Avantor</td>
			<td>82027-402</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Superscript III Reverse Transcriptase (Includes 5x First-Strand Buffer and 0.1M DTT)&#160;</td>
			<td>Invitrogen</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>SUR-VET syringe with needle 25 G x 5/8&#34;, 1 mL</td>
			<td>Terumo</td>
			<td>100281</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green (qPCR enzyme master mixture)</td>
			<td>Applied Biosystems</td>
			<td>A25778</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Manual Slide Staining Set (jars)</td>
			<td>Electron Microscopy Sciences</td>
			<td>SKU: 62540-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Fisher Chemical</td>
			<td>T324-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Avantor</td>
			<td>414004-005</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Chemical</td>
			<td>X3P-1GAL</td>
			<td></td>
		</tr>
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</table>

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

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

1 Our lab is focused on establishing<v>2 supraclavicular brown adipose tissue 3 or SCBAT as a valuable depot 4 for brown fat analysis due to its anatomical similarities 5 to human brown adipose tissue. 6 This protocol is aimed at increasing accessibility<v>7 of SCBAT research. 8 This depot may be intimidating due to its small size 9 and proximity to large veins in the neck, 10 so it is imperative to provide 11 a simple method of extracting and processing SCBAT.12 Our methodology stands out<v>13 firstly by the use of a dissecting microscope 14 when extracting SCBAT. 15 This increases the precision 16 and extracting efficiency, 17 which are both critical when working 18 with such a small tissue depot. 19 Secondly, we homogenized the frozen SCBAT samples 20 before doing gene expression analysis 21 to increase the RNA yield to compensate 22 for such a low quantity of available tissue.23 We are currently focus on defining<v>24 the genetic lining edge of SCBAT, 25 identifying the transfiguration networks 26 regulating its development, 27 and developing a cerebral model 28 for exploring its physiological function in humans.

Unlike iBAT, which is situated in the subcutaneous layer of the back between two scapulae, scBAT is situated in the intermediate layer of the neck, extending deep between layers of skeletal muscle and the salivary gland as it grows along the external jugular vein (Figure 1A). Dissecting scBAT is not as straightforward as iBAT. Here, we provide a detailed procedure including crucial steps for dissecting intact scBAT from postnatal and adult mice (<strong class...

Watch this Scientific Journal Video about Author Spotlight: Optimizing Supraclavicular Brown Adipose Tissue Extraction for Genetic Analysis at JoVE.com

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 ...

dissection-histological-processing-gene-expression-analysis-murine

Research

JoVE Journal

Biology

Dissection of Supraclavicular Brown Adipose Tissue in Mice

1 To begin, place the mouse carcass<v>2 on the work bench. 3 Spray the ventral neck of the mouse with 70%ethanol 4 to minimize fur contamination, 5 make an approximately 1.5 centimeter long bilateral incision 6 along the clavicles. 7 From the ends of bilateral incisions, 8 cut about one centimeter longitudinally towards the ears, 9 creating a squared U-shaped incision around the neck.10 Place the mouse under a dissecting microscope 11 and bring the exposed neck into the focus. 12 Using forceps, lift the incised skin sections 13 to expose the salivary glands. 14 Now using surgical scissors 15 and forceps, disconnect the tissue connecting 16 the salivary glands to the surrounding clavicle area 17 and each other.18 Lift the salivary glands to expose 19 the supraclavicular brown adipose tissue or SC bat depots. 20 Observe the bat along the external jugular vein, 21 which is possibly connected to the underside 22 of the salivary glands. 23 Hold the target adipose tissue with forceps 24 and gently peel it away from the salivary glands.25 After removing adipose tissue, 26 continue detaching the SC bat from the external jugular vein 27 and the neck musculature until the deposes on either side 28 of the neck are fully removed. 29 Inspect the dissected SC bat sample 30 and use forceps to remove any remaining connective tissue.

Supraclavicular Brown Adipose Tissue Processing for Hematoxylin and Eosin Staining

1 Begin by placing<v>2 the glass scintillation vial containing 3 the mouse's scBAT in PBS onto the workbench. 4 Replace the PBS with 10 milliliters of cold, 5 4%paraformaldehyde. 6 Place the vial on a nutator, 7 rocking at four degrees Celsius overnight 8 to fix the scBAT.9 The next day, replace the paraformaldehyde solution 10 with 10 to 15 milliliters of PBS. 11 Place the vial on a nutator 12 and rock for 30 minutes at room temperature. 13 Replace the PBS with 0.85%saline 14 and continue rocking for another 30 minutes.15 Then replace the saline with 10 milliliters 16 of 70%ethanol, 0.85%saline solution. 17 Store the vial in a four degree refrigerator overnight 18 or until ready for paraffin embedding. 19 The next day, remove the 70%ethanol saline mixture 20 from the vial and add 10 to 15 milliliters 21 of 95%dehydrant alcohol.22 Rock the vial on a nutator at room temperature 23 for one hour. 24 After the second wash, add 10 to 15 milliliters 25 of 100%dehydrant alcohol 26 and continue rocking on a nutator for one hour. 27 To clear scBAT for embedding, add 10 milliliters of toluene 28 to the vial and continue rocking on a nutator 29 until scBAT is transparent.30 Then embed the scBAT in paraffin wax. 31 Section the scBAT with a microtome 32 and proceed for hematoxylin and eosin staining. 33 Hematoxylin and eosin staining confirmed 34 that scBAT comprises tissue structures characteristic 35 of brown adipose tissue, 36 including numerous small multilocular adipocytes 37 in both postnatal and adult mice.

Total RNA Isolation from Supraclavicular Brown Adipose Tissue for Gene Expression Analysis

1 After dissecting scBAT<v>2 from the euthanized mouse, 3 immediately put the tissue into a micro centrifuge tube. 4 Poke a hole in the top of the tube with a sterile needle 5 and snap freeze in liquid nitrogen. 6 Soak a pestle and thin spatula in liquid nitrogen.7 Pour the snap frozen tissue sample 8 from the micro centrifuge tube into the mortar. 9 Add a small amount of liquid nitrogen to the mortar 10 and thoroughly grind the tissue with the pestle 11 until completely pulverized. 12 Use the thin spatula to scrape and transfer 13 the pulverized tissue back into the micro centrifuge tube.14 Once all tissue is transferred, 15 add 500 microliters of RNA isolation solution to the tube 16 and sonicate for 30 seconds using a pellet pestle motor. 17 Next, use a syringe to pipette up and down 10 times 18 to further lice the tissue, 19 ensuring no solid particles are visible. 20 Add 250 microliters of chloroform and vortex occasionally 21 during a five minute, room temperature incubation.22 Centrifuge the samples at 21, 000 G for 20 minutes 23 at four degrees Celsius. 24 Transfer the top aqueous layer into a new tube 25 and add an equivalent amount of 70%ethanol. 26 Transfer 500 microliters of the lysate 27 into a binding column.28 Centrifuge at 18, 000 G for 60 seconds 29 and discard the filtrate. 30 For reverse transcription, add 0.5 to one microgram 31 of RNA diluted in DEPC treated water to a PCR tube strip. 32 Then dilute the CDNA to 250 nanograms per microliter 33 using DEPC treated water and set up the quantitative PCR.34 The expression levels of marker genes involved 35 in mediating scBAT function were relatively similar 36 between scBAT and interscapular bat.

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

Morphological and Molecular Characterization of the scBAT in Mice

Summary