Ab84598

Livers were homogenized using a Polytron homogenizer in a Tris-NP40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40) supplemented with a protease inhibitor cocktail (Roche Diagnostics). The homogenates were incubated on ice for 10 min and centrifuged at 10000 g for 10 min to remove tissue debris. Fifty µg of proteins were run on SDS-PAGE mini-gels at the appropriate concentration of acrylamide and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked (1 h at room temperature) with a 5% skim milk in 1×TBST (Tris-buffered saline Tween-20: 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.2% Tween-20) solution and probed with an antibody raised against CYP2E1 (1:1000 dilution, rabbit polyclonal ab84598, (R, H), Abcam, Cambridge, UK) or ß-actin (1:200 dilution, rabbit polyclonal sc-81178 (H, M, R, Hm) Santa Cruz Biotechnology INC, Dallas, TX, USA) overnight at 4 °C. After washing with TBST, blots were incubated at room temperature (1 h) with the appropriate secondary antibody coupled to horseradish peroxidase and washed again. Antibody-bound protein was revealed using the ECL reagent (Thermo Scientific). Films were scanned and analyzed using Image J software. All blots were corrected for loading using ß- actin expression.

The liver sample was taken from the middle lobe, fixed in 10% formalin, standardly prepared for embedding in paraffin, and cut into 5 μm thick paraffin sections. For analysis of iNOS, CYP2E1, SOD1, SOD2, and CAT expression, liver sections were stained immunohistochemically following the Ultravision LP Detection System protocol (TL-125-HD, Thermo Scientific) according to manufacturer’s instructions as described previously [2 (link),29 (link)]. IHC was carried out using an anti-SOD1 antibody (1:1000, ab13498, Abcam), anti-SOD2 antibody (1:200, ab13533, Abcam), anti-CAT antibody (1:1000, ab16731, Abcam), anti-CYP2E1 antibody (1:200, ab84598, Abcam), and anti-iNOS antibody (1:100, ab15323, Abcam).
Digital images of stained sections were taken on a Motic™ B3 Series microscope with Moticam 2500 camera (Motic).