Table 1 Patient (n=44) and tumour (n=45) characteristics For the detection of Fas and FasL by western blotting, six frozen primary GIST samples were pulverised and dissolved in ice-cold PBS. After centrifugation at 18000g for 1min to remove debris, the supernatant was collected. Western blot analysis was performed as described http://www.selleckchem.com/products/Enzastaurin.html above. All tumour samples used in this study were handled according to the guidelines of the Dutch Federation of Biomedical Scientific Societies (FMWV) as described in ��Code Proper Secondary Use of Human Tissue’. Tissue microarray construction Representative regions of the paraffin-embedded primary tumours were selected using H&E-stained slides and arrayed into a tissue microarray (TMA), as described previously (Westra et al, 2005). Briefly, three 0.

6mm tissue cores were taken from (distinct) representative areas of each tumour specimen using a manual tissue arrayer (Beecher Instruments, Sun Prairie, WI, USA) and then transferred to a standard-size recipient paraffin block. The array contained 161 tissue cores including 45 tumour samples in triplicate and 13 normal tissues in duplicate, the latter serving as an internal control for immunohistochemistry. Immunohistochemistry Sections of 4��m were taken from each array block and deparaffinised in xylene. Immunohistochemistry was performed as previously described (Arts et al, 2005). Antigen retrieval was carried out by autoclaving sections in blocking solution (2% blocking reagent (Roche Diagnostics, Mannheim, Germany) and 0.2% SDS in maleic acid buffer (pH 6.0)) at 115��C for three times 5min.

Primary antibodies were mouse anti-Fas (clone CH-11, 1:100; Upstate Biotechnology, Lake Placid, NY, USA) and mouse anti-FasL (clone 33, 1:160; BD Transduction Laboratories). As a negative control, a serial section was processed without the addition of primary antibody. Normal tissue samples within the TMA block derived from liver and kidney served as positive controls for Fas and FasL staining (Leithauser et al, 1993; Lee et al, 1999). Staining analysis Immunohistochemistry results were scored independently by two observers (BR and AJHS) without knowledge of the clinicopathological data. As in each individual core all tumour cells showed the same staining, cores were scored in a semiquantitative manner for staining intensity: no staining (0), weakly positive staining (1), positive staining (2), strong positive staining (3).

Discrepant scoring results were discussed under a multiheaded microscope to achieve consensus. If cores from one tumour differed in staining intensity, the median score of the three related cores determined the score of the tumour. Furthermore, the staining pattern was determined from each core. Statistical Entinostat analysis Data analysis was performed using SPSS 12.0 software package for windows (SPSS Inc., Chicago, IL, USA).