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ELISA for human IL-6 (Pierce-Endogen, Rockford, IL) and MMP-8 (R&D Systems, Minneapolis, MN) were performed in duplicate by investigators unaware of clinical outcomes.
Elemental determinations of Mn were performed using high-resolution inductively coupled plasma mass spectrometry (Finnigan XR ICP-MS).
Inhibition experiments using anti-emmprin blocking antibody (UM-8D6, Ancell Corporation, Bayport, MN) were performed as described previously [ 23].
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Microneutralization (MN) was performed according to the method previously described by Nicholson and colleagues [2].
Human VEGF-C ELISA (R&D Systems, Minneapolis, MN) was performed on medium conditioned by cells according manufacture's instructions.
Staining with a biotinylated anti-NgR antibody (R&D Systems, Minneapolis, MN) was performed similarly, except that the antibody was conjugated to streptavidin PE.
Analysis of intrahepatic IL-6 (Abcam, Cambridge, UK) and OSM (R&D Systems, Minneapolis, MN) was performed in duplicates (n=6) using a murine ELISA kit.
Quantikine mouse TGF-β1 ELISA (R&D systems, Minneapolis, MN) was performed according to the manufacturer's protocol, and the TGF-β1 levels were normalized to those of total protein.
When opinions differed, discussion with a third researcher (MN) was performed to reach consensus and if consensus could not be reached the decision was based on the views of the majority.
To assess the presence of mature IL-1 β, an ELISA (R&D Systems, Inc., Minneapolis, MN) was performed on concentrated CM samples in triplicate according to the manufacturer's protocol.
Immunostainings of EB-MN were performed after fixing samples in 4% paraformaldehyde for 30 min at room temperature.
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