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

Summary

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

Abstract

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.

Introduction

The increasing prevalence of obesity in the U.S. and worldwide has ignited great interest in understanding its etiology and identifying potential treatments^1,2. 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 cold^3,

Protocol

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

1. Place the mouse ca.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
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