Y5200

Tissue microarrays were constructed from 94 tumor cases (45 GC and 49 AEG) using a manual tissue array (Beecher Instruments, Inc., Sun Prairie, WI, USA). For immunohistochemical analysis, 4 µm sections were cut from tissue microarray blocks and tissue blocks just before use. Immunohistochemistry was performed using the Ventana Benchmark XT autostainer (Ventana Medical Systems Inc./Roche Diagnostics, Tucson, AZ, USA), involving a labeled streptavidin-biotin-peroxidase method, followed by visualization with 3,3'-diaminobenzidine.
The primary antibodies used included mouse monoclonal anti-MLH1 (clone ES05, dilution 1:50; Novocastra Laboratories Ltd., Newcastle, UK) and anti-p53 (DO-7, dilution 1:50; Novocastra Laboratories Ltd.). The presence or absence of MLH1 immunostaining was evaluated in the nuclei (Fig. 1f-g). The results of p53 immunohistochemistry were determined as "aberrant" when < 1% of tumor nuclei were positive for p53 (null pattern) or when more than 50% of the neoplastic cells were positive for p53 (overexpression pattern); the remaining results were considered as "wild type." EBV-encoded small RNA in situ hybridization (EBER-ISH) was performed on paraffin sections using a fluorescein isothiocyanate-labeled peptide nucleic acid probe (Y5200; Dako) and anti-FITC (V0403, dilution 1:200; Dako, Glostrup, Denmark).