Supplementary Materials and Methods
Preparation of tissue sections
Biopsy cores were put into RNAlater (Qiagen) and kept at 4° C for tissue preservation and
storage. Before sectioning the samples were washed twice in ice cold PBS for 5 minutes and
incubated in ice cold 30 % (w/v) sucrose in PBS for 1 hour. The biopsies were then embedded in
Tissue-Tek OCT compound (Sakura Finetek Europe) and frozen in isopentane/dry ice slurry. 10
µm sections were prepared using a Leica CM3050 cryostat (Leica Microsystems, Wetzlar,
Germany) set at -25° C and mounted on Superfrost Plus glass slides (Menzel, Braunschweig,
Germany). The slides were briefly warmed to adhere the sections to the glass and then
immediately cooled to -25° C to prevent deterioration of the unfixed sections.
Hematoxylin/Eosin (HE) staining
For fixation slides were immersed in ice-cold acetone for 7 minutes and subsequently hydrated
through a descending ethanol series. Mayer’s Hematoxylin (Histolab, Göteborg, Sweden) and
alcoholic Eosin Y (Sigma-Aldrich) solutions were used according to the manufacturers’
recommendations. Images of HE stained sections were captured on a Nikon Eclipse E800
microscope with a DXM1200 digital camera system (Nikon, Japan).
Immunofluorescence
Sections for immunofluorescence were fixed and permeabilized by incubation in ice-cold acetone
for 7 minutes. After washing in PBS sections were blocked for 1 hour at room temperature in
dilution buffer consisting of 10 % FBS in DMEM (both from Life Technologies, GIBCO) and
incubated with a polyclonal rabbit anti-UCP1 antibody (Sigma U6382, 1:500) in dilution buffer
at 4°C overnight. This was followed by 4x10 min washes in PBS and incubation with Alexa
Fluor 488 conjugated goat anti-rabbit IgG secondary antibody, dilution 1:500, and ToPro-3
nuclear stain, dilution 1:1000, (both from Life Technologies, Molecular Probes) for 2 hours at
room temperature in dilution buffer. After final 4 x 10 minutes washes in PBS, sections were
mounted with ProLong Gold Antifade (Life Technologies, Molecular Probes). Imaging was
performed on a Zeiss LSM 510 Meta laser scanning microscope.
RNA extraction, cDNA synthesis and quantitative real-time PCR
Total RNA was isolated from tissue dissected from four control and four cold exposed rats using the
RNeasy Lipid Tissue Mini Kit (Qiagen), including an on-column DNAse digestion step according to the
manufacturer’s recommendations. The First Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche Applied
Biosciences) was used to synthesize cDNA. Quantitative real-time PCR analyses were performed on a
ViiA7 Real-Time PCR System (Applied Biosystems) using the PowerSYBR master mix (Life Technologies)
and gene specific primers. The standard curve method using the geometric mean of 18s and Ppia for
normalization was used to determine relative gene expression levels.
Supplementary Figure 1
Supplementary Figure 1 Expression of BAT associated genes in BAT depots detected by MRI/DECT.
Quantitative real-time PCR was used to determine the expression levels of Ucp1, Dio2, Ppargc1a, Adrb3
and Prdm16 in interscapular (iBAT), paraaortal (paBAT) and perirenal BAT (prBAT) sampled from rats
kept at either 22°C (N=4) or 4°C (N=4) for five days. In addition control samples of retroperitoneal WAT
(rpWAT) were sampled and included in the analysis. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.