Immunohistochemical staining for CYP2E1, 3-nitrotyrosine (3-NT) protein adducts, 4-HNE protein adducts, collagen α type 1 and smooth muscle actin proteins was performed by using anti-CYP2E1 antibody (a gift from Dr. Jerome Lasker, Hackensack Biomedical Research Institute, Hackensack, NJ), anti-3-NT adducts IgG (Upstate, Lake Placid, NY), anti-4-HNE adducts IgG (Calbiochem, LaJolla CA), anti-collagen α1, and anti-α-smooth muscle actin (SMA, Millipore) IgGs followed by a Broad Spectrum (AEC) Histostain Plus Kit (Invitrogen). DHE fluorescence was assayed by incubating frozen tissue sections of liver with 40 uM DHE (Molecular Probes, Eugene OR) plus 1 mM NADPH applied to the tissue surface for 30 min in a light-protected humid chamber. After rinsing, fluoresecence of the liver section was detected by a fluoresecence microscope. Control sections were incubated with DHE but without NADPH.
