Immunohistochemical staining was performed on the FFPE samples in 5 μm thick, mounted on positively charged slides. Briefly, tissue sections were placed in an oven at 65°C to melt the paraffin, followed by deparaffinization with xylene and rehydrated through serially gradient alcohol solutions. The sections subsequently were immersed into 0.01 M sodium citrate buffer (pH 6.0) and were heated intermittently in a microwave oven for 20 minutes to nonenzymatic antigen retrieval. Endogenous peroxidase activity was blocked with endogenous enzyme block solution (Dako Cytomation Inc., Carpinteria, CA) for 10 minutes.
The primary antibody used to detect specific T-Ag protein expression was a mouse monoclonal antibody against SV40 large T-Ag with cross reacting capacity with JCV T-Ag (clone pAb416, 1:100 dilution; Oncogene Research Products, San Diego, CA). Incubation of the primary antibody was performed overnight at 4°C and was followed by incubation in Dako EnVision+system-HRP polymer (Dako Cytomation Inc.) for 30 minutes. Staining was developed by reaction with diaminobenzide chromogen for 5 to 10 minutes. Next, slides were counterstained for 5 minutes with hematoxylin. The brown chromogen complexes were indicative of T-Ag-specific expression in the gastric tissues.
The primary antibody used to detect specific T-Ag protein expression was a mouse monoclonal antibody against SV40 large T-Ag with cross reacting capacity with JCV T-Ag (clone pAb416, 1:100 dilution; Oncogene Research Products, San Diego, CA). Incubation of the primary antibody was performed overnight at 4°C and was followed by incubation in Dako EnVision+system-HRP polymer (Dako Cytomation Inc.) for 30 minutes. Staining was developed by reaction with diaminobenzide chromogen for 5 to 10 minutes. Next, slides were counterstained for 5 minutes with hematoxylin. The brown chromogen complexes were indicative of T-Ag-specific expression in the gastric tissues.