Immunohistochemical Detection of iNOS

Paraffin-embedded liver sections (4 µm) were used for detection of iNOS. Briefly, sections were deparaffinized and unmasked by heat treatment (Target Retrieval Solution; Dako, Glostrup, Denmark). Sections were stained using either a self-made in-house (24, 25) or a commercially available (Enzo Life Sciences, Lörrach, Germany) rabbit polyclonal antimurine iNOS antibody overnight at 4°C. Notably, both iNOS detecting antibodies generated analogous iNOS staining in livers of APAP-treated mice. For detection, goat antirabbit ABC staining system (Santa Cruz Biotechnology) and the 3,3′-diaminobenzidine Substrate Kit for Peroxidase (Sigma-Aldrich) were used. Sections were counterstained with hematoxylin.