Cellular localization of ErbB-2 in tumor samples. A. IHC staining using RBT-HER2. Representative tumors with 2+ and 3+ scores of membrane ErbB-2 expression (400×). Inset shows FISH of ErbB-2 gene amplification in the tumor scored 2+ (1000×). B. IHC of ErbB-2 (400×) using A0485. Representative tumors showing only membrane ErbB-2 or membrane and nuclear ErbB-2 staining C. IHC of ErbB-2 (400×) using C-18. Examples of tumors showing exclusively membrane, membrane and nuclear or only nuclear positivity. D and E. IF staining of ErbB-2 with C-18. D. Nuclear ErbB-2 score. Nuclei were stained with propidium iodide (red). E. Examples of tumors showing only membrane, membrane and nuclear, and exclusive nuclear ErbB-2 expression (+2 and +3 scores). Nuclei were stained with DAPI (blue). In A to C and in E orange arrows indicate membrane ErbB-2 positivity and in B, C, and E white arrows indicate nuclear ErbB-2 presence. Membrane ErbB-2 levels in both IHC and IF were scored according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines for ErbB-2/HER2 testing . A score 0 represents no staining, 1+ weak, incomplete membrane staining in any proportion of tumor cells, 2+ complete membrane staining that is either non uniform or weak but with obvious circumferential distribution in at least 10% of tumor cells, or invasive tumors showing intense, complete membrane staining in 30% or fewer tumor cells, 3+ strong complete membrane staining in > 30% of tumor cells.