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The following are some of the methods used
in our laboratory to achieve our project goals.
If you have any questions or concerns about
the techniques described here please refer to our Publications for more details.
You can also contact us by E-mail - contact@perinatalreasearch.com
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The Amniochorion Organ Explant
Culture:
Fetal membranes were harvested after the separating from the placenta. Membranes were cleansed of all blood and
decidua using sterile heparinized saline. Using a sterile cotton gauze membranes were further wiped to remove all
the adhering decidua. Membranes were cut into 5 mm circles using a biopsy punch, washed in sterile HBSS x3 and
placed in an organ explant system in media consisting of Dulbecco's Modified Eagles medium (DMEM) : F12 Ham mixture
(1:1) with antibiotics, 15% FBS and 2mM Glutamine (all reagents from Sigma Chemicals, St. Louis, MO). Media and
the fetal bovine serum used in our system contained the lowest endotoxin levels commercially available. Cultures
were incubated at 37oC in an atmosphere of 5% CO2 and room air, and the medium was changed on a daily basis. Microscopic
examination by phase-contrast microscopy was performed frequently and visible evidence of contamination or tissue
death was recorded. Membranes were maintained in organ culture for ten days. During this time viability was confirmed
by active metabolism as documented by continued release of metabolites into the media, constitutive GAPDH mRNA
production, and evidence of viability as determined by phosphatidylcholine test which measured phospholipase activity.
Hematoxylin-Eosin staining done at various time points indicated good tissue morphology. Reverse transcriptase-polymerase
chain reaction (RT-PCR) was performed using specific primers for GAPDH (Glyceraldehyde 3 phosphate dehydrogenase)
on respective samples collected and frozen each day. GAPDH expression was noticed in the fetal membranes throughout
a ten day period in culture. In situ hybridization performed on paraformaldehyde/PBS fixed tissue showed uniform
message for GAPDH in amnion and chorion cells up to ten days. In situ hybridization performed with the sense strand
for GAPDH provided a negative reaction indicating the specificity of the antisense strand for GAPDH mRNA. To study
the response of the membrane in the culture system, tissue samples were stimulated with various doses of endotoxin
(E.coli O55:B5) at the end of a 48 hr incubation period. The 48 hr preincubation time was chosen because genes
of interest (cytokines) were induced by the physical manipulation in preparation for culture. A return to the base
line value of gene expression was achieved over this 48 hr period.
Sigma
Chemical
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Testing of tissue viability
of amniochorionic membrane in culture:
Amniochorionic membrane in culture were tested for tissue viability by looking for expression of message for GAPDH
(house keeping gene). Message expression was studied using RT-PCR (method described in detail below). Hematoxylin-Eosin
staining were performed on respective samples at different time points and the tissue morphology analyzed under
light microscopy. The release of PLC (phospholipase C) and its hydrolytic reaction with PNPC (p-nitrophenylphosphorylcholine)
assayed as an indicator of tissue viability. The method of Sheikhnejad and Srivastava modified for amniochorionic
tissue by Ohno et al were used. Normal and stimulated tissues were collected and stored at -70oC. The tissue was
homogenized with 1 ml of 0.25mol/L Tris.HCl (pH 7.2) and the homogenate will be centrifuged at 1000g for 10min.
Supernatant will be stored at 4oC. The activity of PLC was assayed using PNPC as a substrate. The PNPC solution
(20 mol./L) is made in 0.25 mol/L Tris.HCl pH 7.2 containing 1% BSA (w/v) The assay mixture containing 0.3 ml of
the substrate and 0.15 ml of the supernatant was incubated at 30oC for 30 min, and the absorbance is measured at
405 nm during incubation. The activity of PLC is expressed in U/mg protein (One unit of the enzyme activity equals
1 micromole of the substrate hydrolyzed per minute).68,103
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Reverse transcriptase-polymerase chain
reaction (RT-PCR):
We extract RNA using TRIzol reagent (Superscript Kit, GIBCO-BRL, Grand Island, NY) . DNAse digestions were performed
with RNAse free DNAse and the concentration of the precipitated RNA were measure by spectrophotometry. The expression
patterns of cytokine mRNA were determined by RT-PCR. For the PCR, we use primers from our previous experiments
whose specificity to amplify the required gene sequence has been proven by southern blotting , restriction analysis
and sequencing of the PCR products. All new primers planned to be used in our studies are tested for specificity
using southern blot hybridization, sequencing of the PCR product and by restriction digestion analysis. Total RNA
(0.5 µg) was submitted to random hexamer primed (Superscript Kit, GIBCO-BRL, Grand Island, NY) first strand
cDNA synthesis followed by 30 cycles of PCR in a Perkin-Elmer Cetus thermocycler (Perkin-Elmer Cetus Norwalk, CT).
PCR products were analyzed on 2% agarose gels and visualized using ethidium bromide. A 1kb DNA ladder (GIBCO-BRL)
is used as a molecular weight marker. Representative samples from each batch of experiments were subjected to RT-PCR
without reverse transcriptase enzyme to rule out DNA contamination and possible amplification of genomic DNA.
Perkin-Elmer
Cetus
GIBCO-BRL, Life Technologies
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Quantitative Competitive Polymerase
Chain Reaction:
Quantitative Competitive PCR (QPCR) was achieved using the CLONTECH PCR MIMIC Construction Kit (CLONTECH, Palo
Alto, CA). In this competitive PCR assay one set of primers is used to amplify both the target gene cDNA and another
neutral DNA fragment (MIMIC DNA). The neutral DNA fragment competes with the target cDNA fragment for the same
primers and acts as an internal standard. This protocol uses a non-homologous DNA fragment engineered to contain
the desired gene template primers and thus is recognized by a pair of gene specific primers. Serial dilutions of
the PCR MIMICs are added to PCR amplification reactions containing constant amounts of experimental cDNA samples.
A constant amount of the cDNA is then coamplified with known concentrations of the competitor DNA for 30 cycles.
This MIMIC utilizes the same primer as the target cDNA but yields a PCR product of different size. Following PCR,
the products were resolved by 1.6% agarose gel electrophoresis and visualized on an ethidium bromide stained gel.
A visual comparison is made of the intensities of the bands produced by the target gene and the PCR MIMIC.
Clonetech
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Densitometry of QPCR products:
Quantitation of PCR products was achieved by gel band spot densitometry using the Alpha Ease software program (AlphaInnotech
Corporation, San Leandro, CA). Integrated density values for two matching bands (MIMIC and target genes) were obtained
and the densitometric ratio was calculated. This value was multiplied by the known concentration of the MIMIC to
get the approximate (~) number of molecules. Since the molar concentrations of the MIMIC are known, the relative
levels of expression of target mRNA can be estimated (# fold increase/decrease).
AlphaInnotech
Corporation
Top
Paraffin sections for in situ hybridization
and immunocytochemistry:
6 µm tissue sections were floated on to the surface of chrome-alum gelatin coated slides and fixed at 42oC
overnight. Slides for in situ hybridization was deparaffinized by two, 10 min incubations with xylene and stored
in cold absolute ethanol until processing. For immunocytochemistry, the sections were deparaffinized with 2 changes
of xylene, rehydrated with descending grades of alcohol and equilibrated with PBS pH 7.4.
Fisher
Scientific
Daigger
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In situ hybridization:
In situ hybridization was performed using biotinylated oligonucleotides. The same antisense (3') oligonucleotides
used for PCR were used as the hybridization probe. The sense strand (5') were used as a negative control for the
specificity of hybridization.
The hybridization solution were prepared by mixing 50% v/v deionized formamide, 2x Denhardt's solution, 2x SSC,
0.1% SDS, 0.1 M Tris pH 8.0 and 20 mm vanydyl ribonucleoside complexes (GIBCO-BRL). Slides stored in ethanol were
incubated with hybridization solution and prehybridized in hybridization solution for 1 hr at room temperature.
Biotinylated probes were added to the hybridization solution to a final concentration of 0.5 µg/ml. Hybridization
was done at 450-550C for 16-18 hrs in a humidified chamber followed by washing with 0.1X SSC at room temperature
for 15 min. After blocking endogenous alkaline phosphatase activity, hybridization was detected using streptavidin-alkaline
phosphatase followed by incubation with nitroblue tetrazolium-4-bromo-5-chloro-3- indolylphosphate (In situ hybridization
detection kit, GIBCO-BRL) in alkaline buffer solution pH 9.5. Sections were washed in deionized water and mounted
with Gelmount (Biomedia Corp. Foster City, CA). Photo microscopy of the sections showing reactions will be performed.
Gibco-BRL, Life technologies
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Immuno histochemistry:
Rehydrated sections were quenched in 3% hydrogen peroxide in methanol to block endogenous peroxidase activity followed
by a rinse in PBS and blocked with 10% FBS in PBS for 10 min to minimize nonspecific antibody binding. Sections
were incubated in PBS at room temperature for 30 min in a humidified chamber with primary monoclonal antibody to
the cytokine or substance in question at a dilution to be individually determined for each antibody. After washing,
biotinylated anti-mouse immunoglobulin (Zymed, San Francisco, CA) and avidin-horse radish peroxidase (Zymed) were
added. The chromogenic reaction was developed with a freshly prepared solution of tetra hydrochloride diaminobenzidine
and hydrogen peroxide (Zymed). Sections will be slightly counter stained with Harris hematoxylin. The following
antibodies were normally utilized in our laboratory as controls in each experiment; antibodies to collagen type
IV (Zymed), fibronectin (Zymed), laminin (AMAC, Inc. Westbrook, ME) as a positive control and anti-HLA-DR (DAKO,
Denmark), antibody to single stranded DNA were used as negative control for amnion and chorion cells. PBS without
primary antibody was used as a reagent control. Photo microscopy of the sections showing reactions were performed.
Zymed
laboratories
Calbiochem
OncogeneProducts
Sigma
Chemicals
Biosource
International
Promega
R
& D Systems
Innogenex
Santacruz
Biotechnologies
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Zymography :
Media samples frozen during the experiments were subjected to zymography using specific substrates (gelatin and
casein) for detecting the presence of gelatinases (MMP2 and MMP9). Media containing 0.2% lactalbumin hydrolysate
were used instead of 15% FBS media to avoid any interference from MMPs in FBS. Sodium dodecyl sulfate (SDS), substrate
gel electrophoresis (zymography) was carried out by incorporating an appropriate dilution of substrate into standard
10% polyacrylamide gel electrophoresis (PAGE). After electrophoresis each gel was washed two times for 15 min each
in 2.5% Triton X-100 (to remove SDS) and then incubated overnight in 500 mM Tris.Hcl, pH 7.4, containing 10mM CaCl2.
Each gel was stained with Coomassie blue for 30 min and then destained in 30% methanol-10% acetic acid. Clear bands
on a blue background, due to degradation of the entrapped substrate, indicate the presence of substrate-degrading
proteinases whose molecular weight can be estimated from the molecular weight standards simultaneously run on the
same gel. A second gel run with the same samples were incubated in a buffer containing 10 mM EDTA to document the
metalloproteinase nature of these proteinases.
Novex
Laboratories
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Quantitation of zymograms for active
MMP2:
Human recombinant active MMP2 (Oncogene Research) were used as standard to run zymograms. Different dilutions of
standards (range 1 ng - 0.0001 ng) was made and will be subjected to zymography. The bands produced by these standards
of known concentrations was used to calculate the concentration of active MMP2 (a 66 kDa band) in AF samples, culture
media, and tissue homogenate by densitometry using the Alpha-Ease soft ware program (also see QPCR quantitation).
Oncogene
Research Products
AlphaInnotech
Corporation
Top
Bioactivity testing of MMP9:
The presence of bioactive MMP9 in AF was determined using a bioassay specific for the active form of MMP9 only
(Amersham Life Sciences). The assay uses a urokinase proenzyme that can be cleaved into an active enzyme by a single
proteolytic step. The natural activation sequence in the proenzyme was replaced by protein engineering, with an
artificial sequence recognized only by MMP9. Briefly, MMP9 present in the amniotic fluid was captured onto the
wells by incubating at 4oC overnight in an anti-MMP9 antibody coated micro titer plate. After washing, the plates
will be incubated at 37oC with a substrate protein (an engineered protein which can be cleaved only by active MMP9)
and a detection enzyme for 6 hrs. The resulting color was read at 405 nm using an ELISA plate reader. The concentration
of active MMP9 was calculated by regression analysis. For this, standards (recombinant human pro-MMP9) at different
concentrations are prepared according to manufacturer's instruction. The activation of pro-MMP9 standards to active
MMP9 is achieved by treating them with 1mM p-amino phenyl mercuric acetate (APMA). The sensitivity of the assay
used in our laboratory is 0.125 ng/ml.
Amersham
Pharmacia
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ELISA: Enzyme linked immunosorbent
assay:
The release of cytokines, MMPs and TIMPs of interest into the culture medium can be quantitated using ELISA. The
assay procedure involved a multiple-site two step sandwich immunoassay using oligoclonal antibodies(several monoclonal
antibodies directed against different epitopes of each specific cytokine). These assays were performed in our laboratory
with commercially available kits. Manufacturers instructions were followed for each kit to perform ELISA. Standard
curves are developed using duplicate samples of known quantities of recombinant proteins. Sample concentrations
were determined by relating the absorbance obtained to the standard curve by linear regression analysis. Colorimetric
absorption were read at 450 nm using an Inc Star Automatic microplate reader. Reference filters were chosen based
on the assay performed. The assay reagents are generally obtained from the following sources.
R&D
systems
Promega
corporation
Biosource
International
Amersham
Pharmacia
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LM-PCR for DNA laddering:
Genomic DNA samples were isolated from representative tissue samples using NucleoBond nucleic acid purification
protocols (Clontech Laboratories, Palo Alto, CA). Tissue samples (20 mg) were homogenized and digested in buffer
containing proteinase K (0.25 mg/ml) for 2 hours at 50oC. After purification on an AX anion exchange resin DNA
was precipitated with isopropanol and washed in ethanol for purification. Purified DNA was resuspended in Tris.EDTA
(pH 8.0) buffer by overnight shaking at room temperature. Spectrophotometry was performed to quantitate the DNA.
Genomic DNA was subjected to a ligation reaction with dephosphorylated adaptors
(composed of a 12-mer and a 24-mer) to the ends of the DNA fragments. Human cells undergoing apoptosis were expected
to contain nucleosomal fragments (ladders) with 5'-phosphorylated blunt ends which will bind only to the 24-mer.
The unbound 12-mers were released by heating the reaction. The 5' protruding ends were filled in using thermostable
DNA polymerase. The ligated 24-mer were then used as primers in PCR in which the fragments with adaptors on both
ends were exponentially amplified (Clontech Laboratories, Palo Alto, CA). For this, 0.5µg of genomic DNA
was mixed with dephopsphorylated oligonucleotide. The reaction was performed at 55oC for 10 minutes allowing the
mixture to cool to 10oC over 60 minutes followed by incubation at 10oC for 10 minutes. T4 DNA ligase (200 U) was
added and ligations incubated at 16oC for 12 - 16 hours. Ligated DNA (150 ng) was used for PCR assay (100 µl
volume) with 24 bp linker primer and thermostable DNA polymerase (Clontech Laboratories, Palo Alto, CA). Samples
were amplified for 35 cycles of 1 minute at 94oC and 3 minutes at 72oC and a final extension of 15 minutes at 72oC.
To verify the standard amount of DNA (150 ng) in each sample, ligated DNA was subjected to PCR with En-2 (conserved
single copy gene) primer. The samples were amplified for 26 cycles of PCR; 1 minute 94oC , 1 minute 70oC and 1
minute 72oC. This PCR yielded a product of 290 bp with equal intensities in all samples tested. The LM-PCR products
were then analyzed on an ethidium bromide stained 1.2% agarose gel.
Clontech
Laboratories
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Terminal deoxynucleotidyl transferase
(TdT) mediated biotinylated DNA fragment end labeling:
Amniochorionic membrane fixed in Histochoice was processed in a tissue processor. Paraffin embedded tissues were
applied to chrome-alum gelatin coated slides (6µ thickness) and fixed overnight. Deparaffinization was performed
in xylene and dehydration was performed in graded alcohol. Tissue sections were treated with 20 µg/ml of
proteinase K for 20 minutes at room temperature. Endogenous peroxidase activity was blocked in 3% hydrogen peroxide
in absolute methanol for 5 minutes and slides were washed well in Tris buffered saline. Sections were then incubated
with biotin labeled dNTPs for 1.5 hours catalyzed by terminal deoxynucleotidyl transferase (TdT) (CalBiochem, Cambridge,
MA). After stopping the reaction, sections were treated with streptavidin-horse raddish peroxidase conjugate for
30 minutes and the color was developed using diamniobenzidine, hydrogen peroxide/urea mixture in water (CalBiochem,
Cambridge, MA). The sections were then mounted using Gelmount (Zymed, San Francisco, CA). Controls were carried
out with each experiments in which terminal deoxynucleotidyl transferase was omitted from the labeling reaction.
CalBiochem
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