Expression and localization of GPR91 and GPR99 in murine organs
Cell and Tissue Research
Julia Diehl, Barbara Gries, Uwe Pfeil, Anna Goldenberg, Petra Mermer, Wolfgang Kummer,
Renate Paddenberg
Electronic Supplementary Material
Material and Methods
SDS polyacrylamide gel electrophoresis and Western blotting
Small pieces of frozen organs were homogenized in the 5-fold volume of extraction buffer (7
M urea, 10% glycerol, 10 mM Tris-HCl pH 6.8, 1% SDS, 5 mM dithiothreitol, 0.5 mM
phenylmethylsulphonylfluoride, 1x concentrated Complete Mini Protease Inhibitor Cocktail
(Roche Diagnostics GmbH, Mannheim, Germany)) using a ball mill (Mixer Mill MM300;
Retsch GmbH, Haan, Germany). Protein concentrations of extracts were determined by the
Bio-Rad protein assay (BioRad, Munich, Germany). Per lane, 20 µg extract was applied and
resolved by SDS-12% polyacrylamide electrophoresis. Subsequently, proteins were
transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Schwalbach,
Germany) and incubated overnight at room temperature with rabbit anti-human GPR91
antibody (Cat. No. LS-A3315; MBL/MoBiTec GmbH, Göttingen, Germany or LifeSpan
BioSciences, Eching, Germany; for description and comments on the antibody see Table S1)
diluted 1:2,000 in TRIS-buffered saline (TBS), 0.01% Tween 20, 5% milk powder. Unbound
antibody was removed by three washes with TBS, 0.01% Tween 20 and PVDF membranes
were covered for 1 h with peroxidase-conjugated goat anti–rabbit IgG antibody (Pierce,
Rockford, Illionois, USA) diluted 1:10,000 in TBS, 0.01% Tween 20, 2.5% milk powder.
Finally, bound antibody was visualized by the enhanced chemiluminescent SuperSignal West
Dura Extended Duration Substrate (Pierce). For preabsorption control experiments, rabbit
anti-human GPR91 antibody was mixed with a 100-fold molar excess of the GPR91 peptide
which was used for immunization (Novus Biologicals, Cambridge, United Kingdom). After
24 h incubation at 4°C, the antibody-peptide mixture was applied to the PVDF membrane as
outlined above. For a densitometric evaluation of the signals, X-ray films were scanned and
mean intensities of immunoreactive bands were calculated on a scale of grey values ranging
from 0 to 255 using open-to-public software “ImageJ 1.37V”. For quantification of the
amount of GPR91 protein in different organs, a sample of the same kidney extract was
included in all western blots as reference.
RNA isolation and RT-PCR
Total RNA from most tissues was isolated using RNeasy mini kit (Qiagen, Hilden, Germany)
according to manufacturer’s protocol. RNA from adipose tissue was isolated by phenolchloroform
extraction
using
TRIzol
reagent
(Invitrogen,
Darmstadt,
Germany).
Contaminating genomic DNA was degraded by treatment of total RNA (up to 1 µg) with 1U
DNase I (Invitrogen) for 15 min at 25°C. Messenger RNA was reverse transcribed using
SuperScript II RNase H- Reverse transcriptase (200 U/onset; Invitrogen) and oligo-dT18 (5
µM; MWG-Biotech, Ebersberg, Germany) as primer for 50 min at 42°C. For subsequent
qualitative PCR, 2.5 µl buffer II (100 mM Tris-HCl, 500 mM KCl, pH 8.3), 2 µl MgCl2 (25
mM), 0.75 µl dNTP (10 mM each), 0.25 µl AmpliTaq Gold polymerase (5U/µl; all reagents
from Applied Biosystems, Darmstadt, Germany), and 0.75 µl of each primer (200 µM;
MWG-Biotech) were supplemented with water to a final volume of 25 µl. Cycling conditions
included an initial denaturation at 95°C for 12 min, 40 cycles with 95°C for 20 s, 60°C for 20
s, 72°C for 20 s, and a final extension step at 72°C for 7 min. Real time RT-PCRs were done
with IQ SYBR Green Supermix (Bio-Rad, Munich, Germany) in combination with the ICycler IQ detection system (Bio-Rad).
Tissue preparation for immunohistochemistry
Immunohistochemistry was performed on frozen sections and on paraffin-embedded tissues.
For the preparation of frozen sections, mice were killed by inhalation of an overdose of
isoflurane (Baxter) and exsanguinated by cutting of the abdominal aorta. Organs were
dissected and transferred into Zamboni fixative (2% paraformaldehyde, 15% saturated picric
acid in 0.1 M phosphate buffer, pH 7.4). After 6 h, the organs were rinsed in 0.1 M phosphate
buffer and washed with increasing concentrations of sucrose (9% and 18% in 0.1 M
phosphate buffer). Subsequently, the tissues were embedded in O.C.T.TM Compound (Tissue
Tec; Sakura Finetek Europe B.V., Zoeterwoude, Netherlands) and frozen in liquid nitrogen.
Frozen sections were cut at 10 µm and air-dried before staining. For preparation of paraffin
sections, organs were fixed in situ by perfusion through the left ventricle with Zamboni
fixative and submitted to further immersion fixation in Zamboni fixative for 6 h. After
washing out the fixative, the organs were processed according to standard protocols. Finally,
the organs were paraffin-embedded and serially sectioned at 6-8 µm. Sections were subjected
to immunohistochemistry after deparaffination, rehydration and inhibition of endogenous
peroxidase activity with 0.3% H2O2 in methanol.
H&E staining
Dried 10 µm thick frozen sections and 6-8 µm thick deparaffinized and rehydrated paraffin
sections were used for H&E staining according to standard protocols. Briefly, sections were
washed in H2O, stained for 1-10 min in Mayer's hemalaun stain (Cat. No 1.09249; Merck
Chemicals GmbH, Darmstadt, Germany) diluted 1:2 and differentiated in running tap water
for 10 min. After a short wash in H2O, sections were stained for 2-5 min with Eosin Y
solution 0.5 % in water (Cat. No. X883.1; Carl Roth GmbH & Co. KG, Karlsruhe, Germany).
Then, sections were washed in H2O, dehydrated in ascending alcohol baths followed by xylol
and finally mounted in Eukitt Mounting Medium.