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Eight weeks after bone marrow transplantation, the chimeric mice were stimulated with LPS for 6 h, and the livers were isolated to measure the levels of hepcidin mRNA.
Livers were isolated and fixed in 10% neutral-buffered formalin.
Twelve livers were isolated from Shmt1+/+ or Shmt1−/− mice on a 129SvEv background and placed immediately in cold phosphate buffered saline at 5°C.
The livers were isolated and fixed with 4% paraformaldehyde and 2.5% glutaraldehyde, and then the samples were embedded in epoxy resin (Spurr; EMS).
Livers were isolated and stored at −80°C for pathological observation.
Twenty four hours later (day 29) the livers were isolated from all fish for RNA extraction.
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RNA from SVEC4-10 and HUVEC was isolated with the high pure RNA isolation Kit (Roche Diagnostics) and RNA from nonfibrotic and fibrotic livers was isolated with the high pure RNA tissue Kit (Roche Diagnostics).
RNA from livers was isolated by phenol extraction (Invitrogen, Carlsbad, CA) (n = 10 controls and n = 10 treated mice).
For rescue experiments of liver tissue materials, the DNA of three different livers was isolated three times independently and transformed into bacteria as described above.
DNA from sperm and livers was isolated with the DNA easy kit (Qiagen) according to the manufacturer's protocol and finally dissolved in 25 50 μl TE buffer.
Liver were isolated, snap frozen and stored at -80°C for future analysis.
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