Deparaffinised and rehydrated tissue sections were incubated with the Epitope Retrieval Solution in a hot water bath for 40min at 95�C99��C. Then, the sections were cooled to room temperature for 20min, washed with Tris buffer for 5min, and endogenous peroxidase was blocked with 3% hydrogen selleck inhibitor peroxide for 5min. The primary antibody was rabbit polyclonal antibody to human HER-2, which recognises an intracytoplasmic part of HER-2, and the primary negative control antibody was an immunoglobulin fraction of normal rabbit serum at an equivalent protein concentration to the antibody to HER-2. The sections were washed with TRIS buffer for 5min and incubated with the primary antibody or the primary negative control antibody at room temperature for 30min.
After rewashing with Tris buffer for 5min twice, the primary antibody was detected by incubating at room temperature for 30min using the visualisation reagents, dextran polymer conjugated with horseradish peroxidase and affinity-isolated goat anti-rabbit immunoglobulin. Subsequently, following the rewashing with Tris buffer for 5min twice, diaminobenzidine was added as a visualisation reagent for 10min and sections were counterstained with haematoxylin. EGFR or HER-2 positivity in the IHC analysis was carried out by three observers (YK, FM, and KK). The intensity of reactivity was scored using four categories: negative, no discernible staining or background type staining; 1+, definite cytoplasmic staining and/or equivocal discontinuous membrane staining; 2+, unequivocal membrane staining with moderate intensity; and 3+, strong and complete plasma membrane staining.
Flow cytometry For the analysis of EGFR expression by flow cytometry, mouse anti-human EGFR mAb (DakoCytomation) as the primary mAb and an FITC-conjugated polyclonal rabbit anti-mouse mAb as the secondary mAb (DakoCytomation) were used, and for the analysis of HER-2 expression, a phycoerythrin-labelled anti-HER-2/neu mAb (Becton Dickinson, San Jose, CA, USA) was used. As a negative control for the primary mAb, mouse immunoglobulin G1 mAb (Beckman-Coulter, Miami, FL, USA) was used. Each step of the incubation with mAb was performed at 4��C for 30min. After cells were washed twice in PBS, the stained cells were analysed on a flow cytometer. Fluorescence in situ hybridisation analysis Fluorescence in situ hybridisation (FISH) analysis was carried out using the PathVysion HER-2 DNA Probe Kit (Vysis, Downers Grove, IL, USA).
The HER-2/neu-SpectrumOrange probe contains a DNA sequence specific for the Drug_discovery HER-2 gene locus (17q11.2�Cq12). The chromosome enumeration probe 17 (CEP 17)/SpectrumGreen probe contains alpha-satellite DNA that hybridises to the D17Z1 locus (centromere region of chromosome 17). To determine the copy number for chromosome 17, we used CEP 17 as the control.