0%

ethidium bromide-stained agarose gel and visualized with ultraviolet light. Gels were photographed and the bands were scanned as digital peaks. Areas of the peaks were then calculated in arbitrary units with a digital imaging system (Photo-documentation system, Model IS-1000; Alpha Innotech Co., San Leandro, CA, USA). To evaluate the relative expression levels of target genes in the RT-PCR, the expression value of the normal pooled liver tissues was used as Wnt/beta-catenin inhibitor a normalizing factor and a relative value was calculated for each target gene amplified in the reaction. Non-expression in any of the studied genes was considered if there was a complete absence, or more than a 75% decrease in the intensity of the desired band in comparison to the band of normal pooled liver tissue [24, 25]. Samples were assayed in batches that included both cases and controls. The absence of bands was confirmed by repeating the RT-PCR twice at different days and by consistent presence of β-actin gene amplification

[32]. Immunohistochemistry Protein expression of the studied proteins was assessed using the following monoclonal antibodies Fas (C236), FasL (sc-56103), Bcl-2 (sc-56016), and Bcl-xL (sc-8392) (all from Santa Cruz Biotechnology, Go6983 mouse inc. Germany). Briefly, from each tumor block, a hematoxylin and eosin-stained slide was microscopically examined to confirm the diagnosis and select representative tumor areas. Tissue cores with a diameter of 1.5 mm were punched from the original block and arrayed in triplicate on 2 recipient paraffin blocks. Five μm sections of these tissue array blocks were cut and placed on positive charged slides to be used for IHC analysis. Sections from tissue microarrays were deparaffinized, re-hydrated through a series of graded alcohols, and processed using the

avidin-biotin immunoperoxidase methods. selleck inhibitor Diamino-benzidine was used as a chromogen and Mayer hematoxylin as a nuclear counterstain. Adenosine triphosphate A case of follicular lymphoma was used as a positive control for Bcl-2, Fas and FasL whereas a case of colon cancer was used as a control for Bcl-xL. Results were scored by estimating the percentage of tumor cells showing characteristic cytoplasmic immunostaining for all examined markers [33]. Protein expression was classified compared to normal hepatic tissue samples. Positive expression was further classified according to the level of expression into mild: ≥ 10%- < 25%, moderate: ≥ 25%- < 50% and high expression: ≥ 50% but during statistical analysis they were broadly classified into negative or positive expression.