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 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'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.
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.
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
2. Processing and hematoxylin and eosin (H&E) staining of scBAT (illustrated in Figure 2A)
3. Gene expression analysis of scBAT
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 (Figure 1B,C). After opening the neck using the provided protocol, a thin layer of scBAT can be identified under a dissecting microscope and peeled off from the connected salivary gland and external jugular vein with a pair of forceps (Figure 1D,E). To assess the depot's morphology, freshly isolated scBAT was processed using the provided processing procedure combined with a previously published H&E staining procedure19 (Figure 2A). As shown in Figure 2B, scBAT possesses tissue structures typical of BAT depots and is composed of many small, multilocular adipocytes in healthy postnatal and adult mice. To assess gene expression levels in scBAT, RNA was extracted from isolated scBAT depots using the provided procedures (Figure 3A). Expression levels of genes of interest can then be assessed by standard RT-qPCR methods (Figure 3A). As shown in Figure 3B, the differential expression levels of genes involved in mediating scBAT function, including Pparg, the master regulator of the BAT development; Fabp4 and Glut4, two nutrient transporters; and Ucp1 and Ppargc1a, two genes involved in thermogenesis, can be readily determined. For comparison, RNA was extracted from isolated iBAT, and the expression of the above-listed genes was assessed using the provided procedures as well (Figure 3A). The expression levels of these genes are relatively similar between these two depots. (Figure 3B).
Figure 1: Anatomical location of scBAT and process of its dissection. (A) scBAT's location in the intermediate layer of the neck. (B) The superficial layer of the neck before and after the skin is removed. Scale bar = 250 μm. Dotted yellow lines outline the area that should be exposed after making the U-shaped incision. (C) Image of a mouse carcass placed beneath a dissecting microscope to increase visual clarity during dissection. (D,E) Representative images of the intermediate layer of the neck before and after scBAT is removed from mice aged 3 weeks and 3 months. Scale bar = 250 μm. Dotted yellow lines outline exposed bilateral scBAT depots; n = 2. Abbreviations: scBAT = supraclavicular brown adipose tissue; sfi = superficial layer; sg = salivary gland; tr = trachea; jv = external jugular vein; sm = skeletal muscle. Please click here to view a larger version of this figure.
Figure 2: Processing scBAT for H&E staining. (A) Flowchart illustrating the major steps of scBAT processing for H&E staining. After dissecting the tissue, it is sequentially fixed, dehydrated, embedded, sectioned, and stained. (B) Representative images of H&E-stained scBAT from mice aged 3 weeks and 3 months; n = 3 for each developmental stage; scale bar = 250 µm for lower magnification images; scale bar = 50 µm for higher magnification images. Abbreviations: scBAT = supraclavicular brown adipose tissue; PFA = paraformaldehyde; H&E = hematoxylin and eosin. Please click here to view a larger version of this figure.
Figure 3: Preparation of RNA from scBAT for gene expression analysis. (A) Flowchart illustrating the stages of RNA isolation and RT-qPCR analysis of scBAT gene expression. Due to its small size and soft texture, snap freezing the scBAT depot and grinding it up in liquid nitrogen is crucial to successful RNA isolation. (B) Relative expression of marker genes, including Pparg, Fabp4, Glut4, Ucp1, and Ppargc1a, in scBAT and iBAT isolated from male mice aged 3 months, n = 5. Data are presented as mean ± SEM. 36B4 was used as a housekeeping gene for normalization. Abbreviations: scBAT = supraclavicular brown adipose tissue; RT-qPCR = reverse transcription-quantitative PCR; LN2 = liquid nitrogen. Please click here to view a larger version of this figure.
In this protocol, we present in detail the procedures for dissecting and processing scBAT for H&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 should be taken to avoid puncturing the veins. Excessive bleeding can make it more difficult to locate the scBAT. To process scBAT for H&E staining, we adapted the use of a published protocol19 with slight modifications to include the use of 4% PFA, dehydrant alcohols, and an organic solvent, toluene, for tissue processing as shown in the protocol section. The entire procedure takes ~6 days to complete, and H&E-stained slides can be imaged the next day after completion of staining.
RT-qPCR using RNA as the starting material is the most frequently used method for gene expression analysis in the field of adipose tissue biology. Because scBAT is a relatively small BAT depot compared to iBAT, obtaining sufficient RNA for gene expression can be difficult. To increase RNA yield, we recommend applying sequential lysate preparation steps as outlined in the protocol section, starting by powdering the tissue using a pestle and mortar while frozen. Using this lysate preparation method, we have successfully obtained a high-yield and high-quality RNA sample for gene expression analysis of scBAT from one adult mouse. This lysate preparation method can also be applied to obtain high-quality protein lysates from scBAT for western blotting with the use of protein lysis buffer.
Using mouse iBAT, researchers have gained substantial knowledge regarding BAT function in thermogenesis and metabolism. The recent identification of a few previously unrecognized BAT depots in mice and humans, including scBAT, revealed the need for more studies before we can fully understand the physiological contribution of BAT in mice and adult humans. Particularly, studies illuminating the origins, functions, and involvement of these newly found BAT depots in thermogenesis and regional or whole-body metabolism are warranted.
The authors have nothing to disclose.
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.
95% Dehydrant Alcohol (Flex 95) | Epredia | 8201 | |
100% Dehydrant Alcohol (Flex 100) | Epredia | 8101 | |
96-well PCR plate | Bio-Rad | MLL9601 | |
Aurum Binding Mini Column | Bio-Rad | 7326826 | |
Aurum High Stringency Wash | Bio-Rad | 7326803 | |
Aurum Low Stringency Wash | Bio-Rad | 7326804 | |
Base Molds (for embedding) | Tissue-Tek | 4122 | |
BD PrecisionGlide Needle 21g x 1 1/2" | Becton Dickinson | 305167 | |
C1000 Touch Thermal Cycler | Bio-Rad | 1840148 | |
Capless Microcentrifuge Tubes 2 mL | Fisherbrand | 02-681-453 | |
Centrifuge | Eppendorf | 5430R | |
CFX Opus 96 Real-Time PCR Instrument | Bio-Rad | 12011319 | |
Chloroform | Thermo Scientific Chemicals | 383760010 | |
Cytoseal 60 Low-viscosity mounting medium | Epredia | 83104 | |
DEPC-Treated Water | Ambion | AM 9906 | |
Dissecting Microscope | Nikon | SMZ1500 | |
DNase Dilution Solution | Bio-Rad | 7326805 | |
DNase I | Bio-Rad | 7326828 | |
dNTPs | Invitrogen | 18427013 | |
Elution solution | Bio-Rad | 7326801 | |
EM 400 embedding medium paraffin | Leica Biosystems | 3801320 | |
Eosin Y (0.5% w/v) | RICCA | 2858-16 | |
Formula R Infiltration medium paraffin | Leica Biosystems | 3801470 | |
Genemark Nutator Gyromixer 349 | Bio Express | S-3200-2 | |
Gill #3 Hematoxylin | Sigma-Aldrich | GHS332-1L | |
HCl (for HCL-Ethanol) | Fisher Chemical | A142212 | |
IP VI Embedding Cassettes | Leica Biosystems | 39LC-550-5-L | |
Koptec's Pure Ethanol – 200 Proof (for 70% Ethanol) | Decon Labs | V1001 | |
MgCl2 (25 mM) | Thermo Fisher Scientific | R0971 | |
Microcentrifuge Tubes 1.7 mL | Avantor | 87003-294 | |
Microseal 'B' Seals (adhseive seals) | Bio-Rad | MSB1001 | |
Microtome | Leica Biosystems | RM2245 | |
Molecular Biology Grade Water | Corning | 46-000-CM | |
Mortar Coors Tek | Thomas Scientific | 60310 | |
NaCl (for 0.85% saline) | Fisher Bioreagents | BP358-212 | |
NanoDrop Spectrophotometer | NanoDrop Technologies | ND-1000 UV/Vis | |
Oligo dT | Invitrogen | 18418020 | |
Paraffin Section Flotation Bath | Boekel Scientific | 14792V | |
Paraformaldehyde (PFA) | Sigma-Aldrich | P6148-500G | |
PCR Tube Strip | Avantor | 76318-802 | |
Pestle by Coors Tek | Thomas Scientific | 60311 | |
Pestle Pellet Motor | Kimble | 749540-0000 | |
Phosphate Buffer Saline (PBS) | Sigma-Aldrich | D8537-500ML | |
Precision Model 19 Vacuum Oven | Thermo Fisher Scientific | CAT# 51221162 | |
Primer: 36B4 (forward) 10 μM 5' TGA AGT GCT CGA CAT CAC AGA GCA 3’ |
Chen lab Oligo database | ||
Primer: 36B4 (reverse) 10 μM 5' GCT TGT ACC CAT TGA TGA TGG AGT GT 3’ |
Chen lab Oligo database | ||
Primer: Fabp4 (forward) 10 μM 5’ ACA CCG AGA TTT CCT TCA AAC TG 3’ |
Chen lab Oligo database | ||
Primer: Fabp4 (reverse) 10 μM 5’ CCA TCT AGG GTT ATG ATG CTC TTC A 3’ |
Chen lab Oligo database | ||
Primer: Glut 4 (forward primer) 10 μM 5’ CTG ATT CTG CTG CCC TTC TGT CCT 3’ |
Chen lab Oligo database | ||
Primer: Glut 4 (reverse) 10 μM 5’ GAC ATT GGA CGC TCT CTC TCC AAC TT 3’ |
Chen lab Oligo database | ||
Primer: PPARg (forward) 10 μM 5’ AGG GCG ATC TTG ACA GGA AAG ACA 3’ |
Chen lab Oligo database | ||
Primer: PPARg (reserve) 10 μM 5’ AAA TTC GGA TGG CCA CCT CTT TGC 3’ |
Chen lab Oligo database | ||
Primer: Ppargc1a (reverse) 10 μM 5' ATG TTG CGA CTG CGG TTG TGT ATG 3’ |
Chen lab Oligo database | ||
Primer: Ppargc1a(forward) 10 μM 5' ACG TCC CTG CTC AGA GCT TCT CA 3’ |
Chen lab Oligo database | ||
Primer: Ucp1 (forward) 10 μM 5’ AGC CAC CAC AGA AAG CTT GTC AAC 3’ |
Chen lab Oligo database | ||
Primer: Ucp1 (reverse) 10 μM 5’ ACA GCT TGG TAC GCT TGG GTA CTG 3’ |
Chen lab Oligo database | ||
RNA isolation solution (PureZol) | Bio-Rad | 7326880 | |
RNase Away (surface decontaminant) | Thermo Scientific | 1437535 | |
RNase H | NEB | M0297S | |
Rnase inhibitor (RNase Out) | Invitrogen | 10777019 | |
Scintillation Vial (glass) | Electron Microscopy Sciences | 72632 | |
Slide drying bench | Electrothermal (Cole-Parmer) | MH6616 | |
Stainless staining rack | Electron Microscopy Sciences | 70312-54 | |
Stereo microscope (for embedding) | Olympus | SZ51 | |
Sugical scissors | McKesson | 43-1-104 | |
Superfine point Straight Dissecting Forceps | Avantor | 82027-402 | |
Superfrost Plus Microscope Slides | Fisher Scientific | 12-550-15 | |
Superscript III Reverse Transcriptase (Includes 5x First-Strand Buffer and 0.1M DTT) | Invitrogen | 18080044 | |
SUR-VET syringe with needle 25 G x 5/8", 1 mL | Terumo | 100281 | |
SYBR Green (qPCR enzyme master mixture) | Applied Biosystems | A25778 | |
Tissue-Tek Manual Slide Staining Set (jars) | Electron Microscopy Sciences | SKU: 62540-01 | |
Toluene | Fisher Chemical | T324-1 | |
Transfer pipette | Avantor | 414004-005 | |
Xylene | Fisher Chemical | X3P-1GAL |