An IHC assay was performed for the detection of HER2 protein expression in FFPE tumor tissue specimens and cell lines. The FFPE tissue and cell line blocks were sectioned to a thickness of 4 to 5 μm and mounted on positively charged glass slides. Sections were dehydrated and heat-fixed in a laboratory oven at 60 °C for 1 hour to increase adherence of the tissue section to the glass slide. Sections were incubated with the PATHWAY anti-HER2/neu (4B5) rabbit monoclonal antibody (Ventana Medical Systems, Inc.), followed by incubation with a secondary antibody-horseradish peroxidase conjugate (ultraView Universal DAB Detection Kit, Ventana Medical Systems, Inc.) using the BenchMark ULTRA IHC/ISH System version 12.2 (Ventana Medical Systems, Inc.) per user instructions.
A dual ISH assay was performed to detect HER2 DNA amplification in FFPE tumor specimens that were IHC 2+. Sections were processed using the INFORM HER2 Dual ISH DNA Probe Cocktail, the ultraView SISH DNP Detection Kit, the ultraView Red ISH DIG Detection Kit (Ventana Medical Systems, Inc.), and accessory reagents using the automated BenchMark ULTRA IHC/ISH System version 12.2 (Ventana Medical Systems, Inc.) per user instructions.
A dual ISH assay was performed to detect HER2 DNA amplification in FFPE tumor specimens that were IHC 2+. Sections were processed using the INFORM HER2 Dual ISH DNA Probe Cocktail, the ultraView SISH DNP Detection Kit, the ultraView Red ISH DIG Detection Kit (Ventana Medical Systems, Inc.), and accessory reagents using the automated BenchMark ULTRA IHC/ISH System version 12.2 (Ventana Medical Systems, Inc.) per user instructions.