PEROXIDASE AND DOUBLE IMMUNOFLUORESCENCE STAINING
For all primary antibodies immunostaining was performed using the three-step indirect
immunoperoxidase technique11 and the two-step immunohistochemical procedure with EnVision
(DAKO, Milan, Italy)11. Briefly, after an overnight incubation at 4°C with primary antibodies,
sections were sequentially incubated with the proper secondary and tertiary horseradish peroxidase
(HRP)-labelled antibodies (all from DAKO) (45min). DAKO EnVision polymer was used to
improve immunoreactivity of the rabbit primary antibodies. Immunohistochemical reactions were
developed using 3,3-diaminobenzedine tetrahydrochloride 0.04mg/ml and H2O2 0.01% and
counterstained with Gill’s Hematoxilin N° 2 (Sigma). All the antibodies were diluted in phosphate
buffered saline (PBS, Sigma) 0.1M supplemented with 5% human serum type 0.
Immunuofluorescence was performed using FITC-conjugated anti-rabbit (Santa Cruz Inc.,
goat anti-rabbit clone sc-2012, dilution 1:20), anti-goat (DAKO, rabbit anti-goat, dilution 1:20) and
anti-mouse (DAKO, rabbit anti-mouse, dilution 1:20), and Texas Red-conjugated anti-mouse
(Vector, horse anti-mouse, dilution 1:20). Following an overnight incubation at 4°C of different
primary antibody mixtures, tissue sections were rinsed three times in PBS 0.1M, incubated for
45min with the corresponding secondary antibodies and then mounted in glycerol supplemented
with DABCO 5% (1,4-Diazabicyclo[2.2.2]octane, Sigma-Aldrich) to avoid fluorescence bleaching.
Peroxidase and fluorescent double immunostainings were analysed at Nikon Eclipse E800
microscope. Images were collected using a digital camera (Nikon, Coolpix 995), stored by the
Fotostation 4.5 software (FotoWare) and analysed by the Photoshop 5.0 software (Adobe).
REAL TIME-PCR
RNAs were resuspended in DEPC (Sigma Chemical Co.)-treated water, quantitated at OD
260/280, and their integrity was ascertained by electrophoresis on 1% agarose gel stained with
ethidium bromide. Reverse transcription reaction of total RNAs (1g) was performed with random
hexamers (Applied Biosystems) 100ng, and M-MLV reverse transcriptase (Applied Biosystems) 50
units, in a final volume of 20L at 42ºC for 30min. Real-time PCR was used to quantify the levels
Fabris et al., Angiogenic Factor Expression in Polycystic Liver Diseases
2
of Ang-1 and Ang-2 isoforms and was performed with the LightCycler system (Roche Molecular
Biochemicals). PCR fragments for Ang-1 and Ang-2 isoforms were produced in a 10L reaction
mixture, including 1L of each cDNA, 0.5M of corresponding specific sense and antisense
primers, and 1x buffer provided by QuantiTecttm SYBR Green PCR kit (Qiagen). A 266-bp
fragment
of
cDNA
of
Ang-1
was
amplified
with
the
oligonucleotides:
5´CTGTGCAGATGTATATCAAGC and 5’AGCATGTACTGCCTCTGACT, while for Ang-2 a
297-bp
fragment
of
cDNA
was
amplified
with
the
oligonucleotides
5’GGAAGACAAGCACATCATCC and 5’ATGCCATTTGTGGTGTGTCC, both annealed at
61°C. PCR was initiated by a denaturation step at 95ºC for 10min, followed by 40 cycles with a
95ºC denaturation for 15s, 56-61ºC annealing for 30s, and 72ºC extension for 30s. In each cycle,
fluorescent detection was measured at the end of the 72ºC extension period. As normalizing control
for each sample, amplification of a 174-bp fragment of -actin cDNA was performed with
oligonucleotides 5´AGCCTCGCCTTTGCCGA and 5’CTGGTGCCTGGGGCG with an annealing
temperature of 60ºC. Melting-curve analysis was done at 65 to 95ºC (temperature transition,
0.2ºC/s) with stepwise fluorescence acquisition, readily showing the specificity of the amplification
products from each couple of primers. Standard curves were generated using decimal dilutions of
one of the samples to be analyzed, the relative quantity of each sample was then determined by the
LightCycler software. Amplified cDNAs was ascertained for their expected sizes by electrophoresis
on 1% agarose gel stained with ethidium bromide.
RAT CHOLANGIOCYTE ISOLATION
Twelve Sprague Dawley rats (weighing 150-200g) were used for experiments of
proliferating cell nuclear antigen (PCNA) protein expression following administration of rat
recombinant VEGF164, human recombinant Ang-1 and Ang-2 (R&D Systems, Space, Milan, Italy)
as single agent or mixed solutions. Animals received care according to the principles outlined in the
“Guide for the care and use of laboratory animals” (Natl. Acad. Press, 1996, 7th edition). Rats were
anesthetized with pentobarbital sodium (50mg/Kg body weight). Immuno-magnetic isolation of
Fabris et al., Angiogenic Factor Expression in Polycystic Liver Diseases
3
cholangiocytes was performed as described15, using the OC-2 primary antibody (1:1000) (a kind
gift of Dr. D. Hixson, Brown University, Providence, Rhode Island). Histochemical assays for glutamyltranspeptidase indicated purity values >95% in all used cholangiocyte preparations.
Viability was evaluated by trypan blue exclusion.
PCNA ANALYSIS BY WESTERN BLOT IN ISOLATED RAT CHOLANGIOCYTES
For PCNA Western Blot analysis, after 2hrs incubation with different stimulus, pure
preparations of cholangiocytes were resuspended in lysis buffer and sonicated 3 times (10s bursts).
Proteins were resolved by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis and
transferred
onto
a
nitrocellulose
membrane
(Amersham
Biosciences,
Little
Chalfont,
Buckinghamshire, England). The membrane was blocked using a 3% solution of nonfat dry milk
and then incubated 1h at room temperature with anti-PCNA mouse monoclonal antibody (1:1.000;
Santa Cruz Biotechnology) and anti-actin mouse monoclonal antibody (1:30.000) as primary
antibodies. The membrane was then incubated for 1h with HRP labelled anti-mouse secondary
antibody (1:2.000; Bio-Rad laboratories Hercules, CA). Proteins were visualized using
chemiluminescence (ECL Plus kit; Amersham Biosciences). The intensity of the bands was
determined by scanning video densitometry.