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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.
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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.
Inhibition experiments using anti-emmprin blocking antibody (UM-8D6, Ancell Corporation, Bayport, MN) were performed as described previously [ 23].
Elemental determinations of Mn were performed using high-resolution inductively coupled plasma mass spectrometry (Finnigan XR ICP-MS).
Quantification of MNs was performed relative to the ventricular lumen.
Immunostainings of EB-MN were performed after fixing samples in 4% paraformaldehyde for 30 min at room temperature.
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