The new TNM staging and Edmonson staging of these patients was based on the clinical and pathological diagnosis (Ng et al, 2006). The study protocol was approved by the Institutional www.selleckchem.com/products/AZD2281(Olaparib).html Review Board of the University of Hong Kong. Informed consent was obtained from each patient before operation. Protein expression of Pyk2 and FAK in tumour and adjacent non-tumour liver tissues by immunostaining and Western blot The paraffin sections of the tissue samples were immunochemically stained for Pyk2 and FAK using Dako EnVision? system (Dako, Glostrup, Denmark). In brief, after de-paraffinisation, endogenous peroxidase activity was quenched by immersing the sections for 30min in absolute methanol containing 0.3% H2O2. The sections were processed to unmask the antigens by conventional microwave oven heating in 10mM citric acid buffer (pH 6.

0) for 12min. The sections were then treated with 10% normal goat serum for 30min to reduce the background staining, followed by treatment of appropriate primary antibodies (Pyk2: cytoplasm staining �C H-109, Santa Cruz Biotechnology Inc., 2145 Delaware Avenue, Santa Cruz, CA, USA; Nuclear staining �C Upstate Biotechnology, Charlottesville, VA, USA; FAK: Upstate Biotechnology) at 4��C overnight. After washing, the sections were incubated with EnVision? secondary antibody for 30min at room temperature and then visualised with chromogenic substrate solution for 2min. The slides were examined under light microscope by two independent investigators with the experience of liver pathology.

According to the intensity and area of the staining signalling, the intracellular protein expression of Pyk2 and FAK was classified into higher expression (over 50% of tumour section) and lower expression (less than 50% of tumour section) groups, respectively by an integrated imaging system (MetaMorph Imaging system version 3.0; Universal Imaging Corp, West Chester, PA, USA). Western blot assay was modified from the method described previously (Liang et al, 2003). Proteins from liver tissues were prepared by using urea buffer (8M urea, pH 8.0). Protein extracts were separated by 12% SDS�CPAGE and transferred to PDMF membrane (Millipore, Billerica, MA, USA) according to the standard protocol. After blocking with 5% non-fat milk for 1h, antibody with proper dilution was hybridised with the membrane at 4��C overnight.

The membrane was washed three times with tris buffered saline with tween 20 (TBS/T) each for 10min and incubated with secondary antibody for 1h at room temperature. Protein signal was detected by ECL Plus system (Amersham Biosciences, Piscataway, NJ, USA). The signals were quantified by scanning densitometry (Syngene, Cambridge, UK). Gene expression of Pyk2, FAK, ezrin and fibronectin in tumour tissues by real-time quantitative RT�CPCR Tissue samples were stored Batimastat at ?80��C until total RNA extraction.