Liver sections were stained with haematoxylin and eosin (H&E) for pathological evaluation, as described before (17). Ethanol-induced steatosis was quantified as the percentage of cells containing fatty droplets; five fields (200×) per liver section were examined (one 200× field area contains about 200 cells). Steatosis scoring was recorded as follows: 0, none; 1, <5%; 2, 5–33%; 3, 34–66%; and 4, >67%. Ethanol-induced necroinflammation was quantified as the number of clusters of 5 or more inflammatory cells per mm2; for this, five 200× fields per liver were examined (one 200× field area = 0.95 mm2). Necrosis scoring was recorded as follows: 0, none; 1, <2 foci per mm2; 2, 2–4 foci per mm2; 3, 5–10 foci per mm2; and 4, >10 foci per mm2. The pathologists were unaware of the treatment groups when evaluating the slides. Immunohistochemical staining (IHC) for 4-hydroxyl-nonenal (4-HNE), 3-nitro-tyrosine (3-NT), collagen I, and alpha smooth muscle actin (α-SMA) was performed by using anti-4-HNE, anti-3-NT, anti-collagen I, and anti-α-SMA antibodies (Millipore), followed by the use of a Broad Spectrum (AEC) Histostain-Plus kit (Invitrogen). No staining was observed in the absence of the primary antibody.
