Download Immunohistochemistry and ELISA

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Supporting Materials and Methods
Mice. Mice were maintained at the Specific Pathogen-Free (SPF) unit of the Animal Facility, The
Hebrew University Medical School, under a 12 h light/dark cycle, and provided with food and
water ad libitum. All animal studies were approved by the Hebrew University-Hadassah Medical
School Ethics Review Board.The C57Bl/6 Mdr2-KO (Mdr2-KO/ B6) mice were produced at our
institute by successive backcrosses of the Mdr2-KO/FVB to the C57Bl/6 strain during eight
generations. The Mdr2+/- F1 hybrids of both FVB and B6 strains were produced by breeding of
the Mdr2-KO males with isogenic WT females, and were used as matching controls. Gal-1KO/FVB mice were produced by crossing between homozygous Gal-1-KO/B6 and the
FVB.129P2-Abcb4tm1Bor strain followed by repeated back-crossing of the resulting hybrid and the
parental FVB.129P2-Abcb4tm1Bor strain. After the 7th backcross, the Gal-1+/- / Mdr2-KO hybrid
was crossed with the FVB/NJ strain, and the generated double heterozygotes Gal-1+/- / Mdr2+/were crossed with each other to obtain the Gal-1-KO Mdr2+/+ strain having at least 96.6% of the
FVB/NJ genetic background (further designated Gal-1-KO/FVB).
Harvesting mouse liver tissues. At specified ages, non-fasting male mice were anesthetized with
isoflurane, blood collection was obtained from the peri-orbital sinus, and mice were then
sacrificed by cervical dislocation. When needed, mice were injected with BrdU (i.p., 100mg/kg
body weight, 0.2 ml, B5002, Sigma Aldrich) two hours before euthanasia. Livers were rapidly
excised and weighed; part of the left lobe was fixed in 4% buffered formaldehyde for histology,
and the remaining liver tissue was quickly frozen in liquid nitrogen and stored at -80°C until use.
All mice were operated at the same time interval between 12:00 a.m. and 15:00 p.m. Liver
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tumors having a diameter 0.5 cm or larger were defined as well differentiated HCC by a qualified
pathologist (O.P.).
Blood analysis. Serum was obtained by centrifugation of total blood at 3,200 g for 10 min.
Hepatocyte damage was assessed for the activities of ALT and ALP (U/L) in the serum by an
enzyme assay using Reflotron (Roche, Mannheim, Germany). Cholesterol level in the serum was
evaluated by Reflotron strips with the measuring range of 100-500 mg/dL.
Genotyping. DNA from mouse tails was purified by D-TailTM Extraction kit (Synthezza Bioscience Ltd, Jerusalem, Israel). Genotyping was carried out by PCR analysis of DNA extracted
from mouse tails as a template using primers specific to genes Mdr2, Neo (1) and Gal-1 (2).
Genome scale gene expression analysis. Gene expression profiling was performed using
Affymetrix (Santa Clara, CA) Mouse Genome Array 430A (22,000 genes). GCRMA
normalization was applied to the raw data. An unpaired T-test was used to find significant
changes in expression patterns between different groups of mice. Expression analysis was
performed using using the Partek software (ProSoftware, Partek, St Louis, MO, USA); GO
enrichment analysis of discovered functional gene groups was performed using the GenMapp and
EASE online tools.
Immunohistochemistry and counting of positive cells.
Immunostaining was done on 4-μm-thick formalin-fixed paraffin-embedded liver tissue sections
by standard procedures. The following antibodies to specific markers were used for
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immunohistochemial stainings: for DNA synthesis, to BrdU (1:50, M0744, clone Bu20a, Dako,
Denmark), for macrophages/monocytes, to F4/80 (1:300, Serotec, Oxford, UK), for T cells, to
CD3 (1:200, MCA1477, Serotec, Oxford, UK), for neutrophils, to Ly6B.2 (1:3000, MCA771GA,
Serotec, Oxford, UK), for lipin-1 (1:100, SC-98450), for Mbd1 (1:100, Santa-Cruz, CA, USA).
Antibody for cyclin D1 was described previously [Katzenellenbogen, 2007 #1046]; antibody to
galectin-1 (1:900) was produced in the laboratory of Gabriel A. Rabinovich. Antigen retrieval
was done with citrate buffer (pH 6.0; Gal-1, Lpin1, Mbd1, CD3, and BrdU), or glycine buffer
(pH 9.0; cyclin D1) in a microwave (Electrolux, Stockholm, Sweden) by boiling (Gal-1, CD3,
Ly6B, BrdU), or using a pressure cooker (cyclin D1, Lpin1, Mbd1). In the case of F4/80, the
antigen unmasking was performed with 1 mg/ml protease XXIV (P8038, Sigma) for 10 min at
R.T. Cas-Block™ (008120, Invitrogen, Carlsbad, CA) was used for dilution of all antibodies as
well as for tissue blocking. The following secondary antibodies were used: anti-rabbit (K4003),
anti-mouse (K4001; both Envision, Dako, Denmark) and anti-goat (414311, N-Histofine,
Nichirei Biosciences Inc., Tokyo, Japan). Color was developed using either 3-amino-9ethylcarbazole (AEC, 00-1111, Invitrogen) for 10 min (30 min in the case of CD3) (Gal-1, CD3,
Ly6B, BrdU) or 3,3’-diaminobenzidene for 5 min (cyclin D1, F4/80). Negative controls were
used by omitting the primary antibody, or using a Gal-1-KO liver in the case of Gal-1. The
stainings were visualized with the Nikon Eclipse E600 microscope equipped with Soft Imaging
System Cell^A. For histological assessment, clinical pathologist (O.P.) evaluated slides in a blind
fashion. The number of F4/80+, CD3+, and Ly6B+-expressing cells for each slide was calculated
as the sum in the x20 or x40 microscope power field. Ten fields per slide were included, and a
total of 4 or 5 mice per group were used. In the case of cyclinD1-positive or Brdu-positive cells,
only hepatocytes were evaluated. Nuclear DNA fragmentation on slides of formalin-fixed,
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paraffin-embedded mouse liver tissues was tested using the In Situ Cell Death Detection Kit
(Roche, Mannheim, Germany).
Immunoblotting. Samples from the whole tissue lysates (50 µg protein/lane) were resolved on
SDS-PAGE (Hoefer Mighty small SE245, Pharmacia Biotech, USA ) and electrophoretically
transferred (Trans-Blot Semi-dry, BioRad) onto PVDF membranes (Bio-Rad, CA, USA). Gal-1
and Mat1a proteins were detected with the above mentioned rabbit anti-Gal-1 (1:5000) and goat
anti-Mat1a (1: 200, Santa-Cruz, CA, USA). Mouse anti-CoxIV (1:200, Abcam, UK) was used as
a control to monitor sample loading. The immune-complexes were visualized using the anti-rabbit
(1:300), or anti-mouse (1:200) antibodies described above, or the donkey anti-goat antibody
(1:14000, Jackson ImmunoResearch, West Grove, PA, USA) for 45 min and the EZ-ECL
detection system (Biological Industries, Beit Ha-Emek, Israel) for 3 min.
Confirmation of differential gene expression by RT–PCR.
Reverse transcription of total RNA was performed using the MMLV Reverse Transcriptase and
random hexamer primers (Promega, Madison, WI). Semi-quantitative PCR was performed using
2xKapa2GFast Taq Polymerase (KM5101, Kappa Biosciences, Norway). For each gene, the
cDNA concentration and the number of PCR cycles were established in the linear amplification
range. Expression levels for each gene were normalized against housekeeping genes Mrps21 or
Hprt. The primers used are shown in Supporting Table 1. Pictures of agarose gels were taken by
B.I.S. 202D BioImaging System (Amersham Pharmacia Biotech., USA). The intensities of bands
were evaluated using the Scion Image program (Scion Corp., Frederick MD, NIH).
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Statistical Analysis. Statistical significance between groups was estimated using two-tailed
unpaired ttest. Results are expressed as a mean ±standard error of the mean (SEM); differences
were considered significant at P <0.05.
References
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Poirier F, Robertson EJ. Normal development of mice carrying a null mutation in the gene
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