Supersensitive streptavidin biotin peroxidase complex

IHC was performed on 10% neutral buffered formalin-fixed, paraffin-embedded (FFPE) tissue. Sections (5-μm thickness) were cut from FFPE tumor blocks and routinely deparaffinized and rehydrated. Anti-HER2 (monoclonal, Thermo Fisher Scientific, Grand Island, NY) at a dilution of 1:100 was used to detect the expression of HER2 on fresh D2F2, D2F2/E2, 4T1, and HER2-4T1 tumors by IHC. HER2 was visualized using the supersensitive streptavidin-biotin-peroxidase complex (Biogenex, Fremont, CA). For antigen retrieval, slides were heated in a microwave oven for 30 min in citrate buffer solution (pH=7.4) and cooled slowly to room temperature. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 8 min. Sections were then incubated with the anti-HER2 antibody overnight (>12 h). Slides were subsequently rinsed in phosphate-buffered saline (PBS) three times and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. After incubation, slides were washed again with PBS and visualized using diaminobenzidine. Mayer’s hematoxylin was used to counterstain sections, which were dehydrated and mounted.