paraffin-embedded sections of liver specimens were obtained from the Departments of Pathology at Beth Israel Medical Center, New York, United States, Kurume University School of Medicine, Kurume, Japan, Aristotle University Medical School, Thessaloniki, Greece, and from the Liver Cancer Specimen Bank, part of the National Research Resource Bank Program, which is administered by the Korea Science and Engineering Foundation under the Ministry of Science and Technology. Approvals from the respective institutional review boards or the equivalent were obtained prior to beginning all investigations. selleck chemical The liver biopsy specimens consisted of 33 cases of chronic hepatitis B (CHB) and 69 cases of chronic hepatitis C (CHC). Histologically normal (control) liver specimens were obtained from wedge-biopsied livers of donors for liver transplantation, autopsy, or normal tissue distant from tumor in hepatic resections. The liver biopsy specimens with chronic hepatitis were staged for fibrosis according to a modified Ishak
staging system19 (1, portal fibrosis; 2, fibrous septa; 3, transition to cirrhosis; 4, established cirrhosis) and for grade of necroinflammatory activity (1, mild; 2, moderate; 3, selleck chemicals severe [i.e., with confluent necrosis]). Four-micron thick tissue sections were deparaffinized with xylene and rehydrated with graded alcohols. After washing in distilled water, sections were immersed in 3% hydrogen
peroxide to block endogenous peroxidase. Details of EpCAM staining methods used at the three institutions are given in Table 1. Other antibodies used for immunohistochemical stains included: keratin (K) 19 (clone RCK108, Dako, Glostrup, Denmark; dilution 1:20), p21WAF1/Cip1 (clone SX118, Dako; dilution 1:50), and 上海皓元 proliferating cell nuclear antigen (PCNA) (clone PC10, Dako; dilution 1:75). These stains were either performed in sequential cuts of the tissue block (EpCAM/K19) or in the same slide (double staining of EpCAM with K19, p21WAF1/Cip1, or PCNA). We used the DAKO Envision Kit (Dako) for immunohistochemistry with a single primary antibody, using 3,3-diaminobenzidine (Dako) as the chromagen. All slides were counterstained with hematoxylin. For double immunohistochemical staining, the EnVision AP system (Dako) and Vector Blue Alkaline Phosphatase Substrate Kit III (SK-5300, Vector Laboratories, Burlingame, CA) were used to detect the first primary antibody, and then the EnVision DuoFLEX Doublestain System (SK110) (Dako) and Vector NovaRED Substrate Kit (SK-4800, Vector Laboratories) were used to detect the second primary antibody.