Ventana benchmark automated immunostainer

One to two representative formalin fixed paraffin tissue blocks containing tumor from each case were retrieved to generate 4 µm thick unstained slides for immunohistochemical staining for SATB2 (dilution 1:100, clone EPNCIR 130A, Abcam, Cambridge, MA) and CDX2 (prediluted, clone EPR2764Y, Cell Marque, Rocklin, CA) on a Ventana Benchmark automated immunostainer (Ventana Medical Systems, Inc., Tucson, AZ) according to standard protocols with appropriate positive and negative controls. Chromogranin (prediluted, clone LK2H10, Ventana, Tucson, AZ) and synaptophysin (predilute, polyclonal, Cell Marque, Rocklin, CA) were also performed for AdexGCCs using a Ventana Benchmark automated immunostainer.
Only nuclear staining was considered positive for SATB2 and CDX2. The staining was reviewed in a consensus manner (CY, LZ, DC) with a multiheaded microscope. The results were scored in a semi-quantitative pattern as 0 (< 1% cells), 1+ (1-25%), 2+ (26-50%), 3+ (51-75%), 4+ (76-100%). The percentage of positive cells was visually estimated with 5% increment.

Clinical GC tissue samples were obtained from patients undergoing gastrectomy at Tokyo University Hospital, with written informed consents. Formalin‐fixed, paraffin‐embedded tissue blocks of EBV(−) and EBV(+) GC were cut into 4‐μm‐thick sections. Three serial sections were prepared for each tissue block and used for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) using anti‐EHF (Abcom, ab167264) and anti‐p‐STAT3 (SAB4300033‐100UG, rabbit polyclonal) antibodies. IHC was performed using Ventana BenchMark automated immunostainer (Ventana Medical Systems), and visualized with OptiView DAB IHC Detection Kit (760‐700; Roche) according to the manufacturer's instructions. Two independent experienced pathologists scored the staining as follows: 3 (strong), 2 (moderate), 1 (weak), and 0 (negative). The study design was approved by the Institutional Review Board of Chiba University and the University of Tokyo.