An increase in TGFβ1 expression in periportal region also appears to be important for the shift from hepatocytic to biliary cellular profile. Methods Materials Collagenase for hepatocyte isolation was obtained from Boehringer Mannheim (Mannheim, Germany). General reagents and 4,4′-Methylenedianiline (DAPM) were obtained from Sigma Chemical RG-7204 Co. (St. Louis, MO). Primary antibodies used are: CK19 (Dako Corp; 1:100), HNF4α (Santa Cruz; 1:50), HNF6 (Santa Cruz; 1:50), HNF1β (Santa Cruz; 1:100), TGFβ1 (Santa Cruz; 1:200). Biotinylated secondary antibodies were obtained from Jackson Laboratories.

Target retrieval solution was obtained from Dako Corp. ABC kit and diaminobenzidine (DAB) kit were from Vector Laboratories. Animals DPPIV positive Fisher 344 male rats were obtained from Charles River Laboratories

(Frederick, MD). DPPIV negative Fisher 344 male rats were obtained from Harlan (Indianapolis, IN). The animal husbandry and all procedures performed on the rats employed for these studies were approved under the IACUC protocol #0507596B-2 and conducted according to National Institute of Health guidelines. Generation of rats with chimeric livers DPPIV chimeric livers were generated as previously described [3, 21]. Briefly, male DPPIV negative Fisher rats (200 g) were given two intraperitoneal injections of retrorsine (30 mg/kg), Forskolin dissolved in water. The injections were given 15 days apart. A month after the last injection, the rats were subjected

Pirfenidone cost to PHx. During the PHx operation, the rats were also injected directly into the portal circulation (via a peripheral branch of the superior mesenteric vein) with 3.5 million hepatocytes isolated from DPPIV positive male Fisher rats (200 g). The animals were left to recover and were not subjected to any other experimental procedures for the next 3 months. Assessment of the degree of engraftment was made under direct microscopic observation of sections from the chimeric livers, stained for DPPIV. The percentage of DPPIV positive and negative cells was estimated at 40× magnification in optic fields that included at least one portal triad and one central vein. The percentage of DPPIV-positive cells varied from one lobule to another. The range of engraftment per optic field (as defined above) within each animal varied from 30% to 60%. Treatment with DAPM Biliary toxicant DAPM (50 mg/kg, dissolved in DMSO at a concentration of 50 mg/ml) was injected intraperitoneally to either DPPIV chimeric or DPPIV positive male Fisher 344 rats every 2 days. In the pilot study, bile duct injury after single injection of DAPM was at its peak at 24 and 48 h after treatment (Figure 1A, B) while PCNA analysis indicated that the biliary cells begin cell division at 48 h (Figure 1C).