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Immunohistochemistry was performed on OCT-preserved mouse heart samples.
Whilst the cDNA prepared from positive control EMC virus RNA, amplified with both primer sets, the cDNA prepared from RNA extracted from mouse heart samples failed to amplify with either of the Cardiovirus primer sets.
We attempted to detect mutant PPARγ allele in heterozygous mouse heart samples to confirm the presence of mutated allele.
Briefly, mouse heart samples were homogenized in 10% homogenization buffer and centrifuged at 1500 g for 5 minutes at 4°C.
The expression of IL-1β in mouse heart samples was determined using a mouse IL-1β/IL-1F2 immunoassay kit (R&D Systems, Minneapolis, MN) and has been normalized to the protein content.
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In Thai patients with infective endocarditis, the mean period of time from symptoms to diagnosis was 5.7 weeks [ 41], and our present study only lasted to 6 weeks, with mouse hearts sampled at weeks 4 and 5 only.
Negative controls (water during the amplification step and an uninfected mouse heart tissue sample during the extraction protocol) were included every five samples for each experiment to monitor potential contamination.
The cDNAs were produced with a First Strand cDNA Synthesis Kit (MBI Fermentas, Heidelberg, Germany) using total RNA isolated from mouse heart and liver samples.
The fifteen mouse heart tissue cRNA samples were labeled with Cy5 and the Universal Mouse reference cRNA was labeled with Cy3, yielding a total of 5 biological replicates per condition.
Briefly, mouse heart and liver samples were homogenized in 10% homogenization buffer and centrifuged at 1323 g for 5 minutes at 4°C.
Mouse heart and skeletal muscle samples were taken from C57BL/6 or CD1 mice purchased from Jackson laboratory.
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