Ventana inform dual ish her2 kit

Paraffin blocks were stored in an environment with controlled temperature and humidity. For IHC, 4 μm sections were cut from each tissue block and stained with hematoxylin and eosin (HE). HE-stained slides were evaluated to select representative tumors and to verify high tumor content. The following antibodies were used for IHC: ER (SP1, rabbit monoclonal primary antibody CONFIRM, Roche Diagnostics, Tokyo, Japan), PgR (1E2, rabbit monoclonal primary antibody CONFIRM, Roche Diagnostics, HER2 (Dako HercepTest, Agilent Technologies, Tokyo, Japan), Ki-67 (MIB-1, Dako monoclonal mouse antibody, Agilent Technologies, Tokyo, Japan). Expression levels of ER and PgR were recorded using the Allred score [35 (link)]. Tumors scoring 3 or higher (corresponding to at least 1% positive cells) were considered positive for ER and PgR [17 (link), 36 (link)]. HER2 status was considered positive if membranous immunostaining was IHC 3+ or IHC 2+ and HER2 gene amplification was confirmed by dual color in situ hybridization using the Ventana Inform Dual ISH HER2 kit (Roche Diagnostics. Tokyo, Japan) [18 (link)]. The Ki-67 labeling index (LI) was calculated by dividing the number of Ki-67–positive tumor cells by the total number of tumor cells. High- and low-LI groups were separated using the mean Ki-67 LI as a cutoff [37 (link)].