This treatment was continued for total 3 times and the rats were sacrificed at day 30 after the last DAPM injection (Figure 2A). The livers were harvested and utilized for DPPIV histochemistry. Additional two groups of normal rats ware given either intraperitoneal injection of 50 mg DAPM/kg every two days for 3 times (DAPM × 3) or single DAPM injection (50 mg DAPM/kg) two days before the bile duct ligation (DAPM+BDL). At the end of 30 days after the
last treatment, rats were sacrificed Blood was collected for serum analysis. Livers were harvested for further analysis. Bile duct ligation Bile duct ligation was performed as previously described . Briefly, the animals were subjected to a mid-abdominal incision 3 cm long, under general anesthesia. The common bile duct was ligated in two adjacent positions approximately Z-DEVD-FMK 1 cm from the porta hepatis. The duct was then severed by incision between the two sites of ligation. Immunohistochemistry Paraffin-embedded liver sections (4 μm thick) were used for immunohistochemical staining. For HNF4α and HNF6 staining, antigen retrieval was achieved by steaming the slides 60 minutes in preheated target retrieval solution (Dako Corporation). For CK19 staining the slides were steamed for 20 minutes in high pH
target retrieval solution (Dako Corporation) before blocking. For TGFβ1 staining no antigen retrieval was necessary. The tissue sections were blocked in blue blocker for 20 minutes followed by incubation with pertinent primary antibody
overnight at 4°C. The primary antibody was then linked to biotinylated secondary antibody followed by routine avidin-biotin complex selleckchem method. Diaminobenzidine was used as the chromogen, which resulted in a brown reaction product. Electronic supplementary material Additional file 1: Serum ALT levels in F344 rats. Serum ALT levels after DAPM (50 mg/kg) administration in F344 rats over a time course, where * indicates statistical difference from the 0h control (P ≤ 0.05). (TIFF 3 MB) Additional file 2: HNF6 immunohistochemistry on liver sections. (A) normal control rats (NRL, normal rat liver), (B) rats that underwent P-type ATPase DAPM + BDL treatment, or (C) repeated DAPM treatment (DAPM × 3). Brown nuclear staining indicates HNF6 positive staining. No appreciable variation in HNF6 expression was noticed in the treatment versus control groups. Scale bar = 100 μm. (TIFF 3 MB) References 1. Michalopoulos GK, Bowen WC, Mule K, Stolz DB: Histological organization in hepatocyte organoid cultures. Am J Pathol 2001, 159:1877–1887.CrossRefPubMed 2. Michalopoulos GK, Bowen WC, Mulè K, Lopez-Talavera JC, Mars W: Hepatocytes undergo phenotypic transformation to biliary epithelium in organoid cultures. Hepatology 2002, 36:278–283.CrossRefPubMed 3. Michalopoulos GK, Barua L, Bowen WC: Transdifferentiation of rat hepatocytes into biliary cells after bile duct ligation and toxic biliary find more injury. Hepatology 2005, 41:535–544.CrossRefPubMed 4.