In addition to control Belinostat 414864-00-9 samples of normal liver, we also examined cases of steatohepatitis and HCC as non-viral disease controls. This gave a larger pool of cases for comparison of different methods of apoptosis quantification. Apoptotic rates were assessed by using H&E morphology and immunohistochemistry for activated caspase-3 and the monoclonal antibody M30. Materials and methods Case material This is a retrospective study using archival formalin-fixed and paraffin-embedded tissue. Liver biopsies and resections were retrieved from the archive and anonymized according to local Ethical Committee guidelines. There were 32 cases of chronic viral hepatitis, including 26 from patients with hepatitis C virus infection, four from patients with hepatitis B virus infection and two from patients with both hepatitis B and C virus infection.
Seven cases of HCC and six of steatohepatitis were used as non-viral disease controls. In addition, blocks of background normal liver from eight liver resections for metastatic adenocarcinoma were selected as control material. Immunohistochemical procedures Formalin-fixed, paraffin-embedded sections were cut to 4 ��m thickness, dewaxed in xylene and rehydrated through graded alcohol to distilled water. The sections were subjected to microwave antigen retrieval for 14 min in 10 mM citrate buffer (pH 6.0). The indirect alkaline phosphatase method was used for activated caspase-3 detection.
The primary antibody (Affinity-purified Rabbit Anti-human/mouse Caspase-3 Active, R&D systems, Minneapolis, MN, USA) was applied at a dilution of 1 : 1000 after normal goat serum, incubated overnight at 4 ��C, then treated with goat anti-mouse/rabbit alkaline phosphatase conjugate (N series ready to use, Dako Ltd, Ely, Cambridgeshire, UK) for 15 min. The alkaline phosphatase anti-alkaline phosphatase (APAAP) method was used for M30 detection. The primary antibody (M30 Cytodeath mouse monoclonal antibody, Roche, Basel, Switzerland) was applied at a dilution of 1 : 50 after normal rabbit serum, incubated overnight at 4 ��C, then treated with rabbit anti-mouse immunoglobulin (Dako P314) at a dilution of 1 : 50 and then APAAP at a dilution of 1 : 100 (Dako D0651). The slides were immersed in naphthol phosphate/fast red substrate for 35 min to demonstrate alkaline phosphatase activity. A negative control reaction with no primary antibody was carried out for every case.
Quantification of apoptosis Sections were examined by light microscopy using a high-power objective, and GSK-3 apoptotic cells in a parenchymal location were counted. On H&E-stained sections, apoptotic cells were identified by their characteristic features of nuclear and cytoplasmic condensation. For sections stained with antibodies to activated caspase-3 and M30, whole-cell cytoplasmic staining was counted; any weak background granular staining was ignored.