Clone 2a8

Liver tissue was fixed in 10 % formaldehyde (Sigma-Aldrich), dehydrated, embedded in paraffin blocks, and sectioned into 5 μm slices. The sections were stained with hematoxylin and eosin (H&E; Sigma-Aldrich) for histology, and the necrosis grade was evaluated in 20 random 100 × images per animal, as described by Liu et al. [11 (link)] as follows: “0” indicated normal; “1” indicated necrotic cells in the first cell layer adjacent to the central vein; “2” indicated necrotic cells extending two to three cell layers from the central vein; “3” indicated necrotic cells extending three to six layers from the central vein; “4” indicated necrotic cells extending three to six layers and from one central vein to another; and “5” indicated necrotic cells throughout the section. The sections were deparaffinized and dehydrated for immunohistology, and the endogenous peroxide was inactivated with 3 % hydrogen peroxidase (Sigma-Aldrich). The sections were then blocked with 3 % normal goat serum (DAKO, Glostrup, Denmark) for 1 h, stained with primary antibodies against cytochrome P450 subfamily 2E1 (1:200), 4-hydroxynonenal (1:200, Abcam, Cambridge, MA, USA), or nitrotyrosine (1:50, clone 2A8.2, Millipore, Bedford, MA, USA) for 1 h at 37 °C and then incubated with an horseradish peroxidase (HRP)-detection kit (REAL^TM EnVision, DAKO) according to the manufacturer’s protocol.