NASH model

Seven-week-old male C57BL/6 mice were divided into two groups. One group was fed a choline deficient diet supplemented with 0.1% methionine on a high fat diet background (CDAHFD) (A06071302, Research Diets, New Brunswick, NJ, USA) for 8 weeks. The second group was fed a CDAHFD and intraperitoneally injected with 35B6 twice a week for 8 weeks. After 8 weeks, blood samples were collected for ALT and OPN ELISA, and livers were removed for HE and Sirius-red staining. Collagen content was quantified using ImageJ Software by assessment of the area of liver tissue that was stained positively by Sirius-red. For each tissue, data were obtained using six high power fields (×100). The areas were quantified in a double-blind manner. The expression of collagen I was assessed by anti-collagen I antibody ab34710 (Abcam, Cambridge, MA, USA). Sections of paraffin-embedded liver tissues were processed for immunohistochemistry according to the manufacturer’s instructions.

Evaluation of the extent of resultant NASH was scored using scaling scores (Brunt et al. 1999). Briefly, steatosis was graded according to the following scale: 0 = normal; 1 = < 33% affected hepatocytes; 2 = 33%–66% affected hepatocytes; and 3 = > 66% affected hepatocytes. Ballooning was graded a score of 0–3 based on severity: 0 = none; 1 = rare or few; 2 = moderate; and 3 = many. Lobular inflammation was graded a score of 0–3 based on inflammatory foci per ×20 field: 0 = none; 1 = 2 foci; 2 = 7 foci; and 3 = > 4 foci/×20 field.