Anti her2 neu

Tumor tissues were fixed in 10% formalin and embedded in paraffin. Immunohistochemical staining was carried out using anti-HER2/neu (Dako, Glostrup, Denmark) as the primary antibody for c-erbB-2. After using a microtome to cut 4 µm-thick tissue sections, the sections were immersed in xylene solution to remove residual paraffin and hydrated in an alcohol series. Sections were boiled for 5 minutes in citrate buffer (pH 6.0) to retrieve antigenicity and left for 30 minutes at room temperature. After exhausting endogenous peroxidase for 10 minutes with H₂O₂ in methyl alcohol, sections were washed thrice with phosphate-buffered saline (PBS). Under room temperature conditions, sections were blocked for 30 minutes with blocking solution (Histostain™ kit; Zymed Company, San Francisco, CA, USA), and then incubated with anti-HER2/neu (1 : 200; Dako). After rinsing thrice with PBS, sections were incubated with biotinylated anti-mouse immunoglobulin G (1 : 300; Zymed Company), washed, and incubated with avidin-alkaline phosphatase for 7 minutes at 40℃. Next, sections were visualized with red chromogen at 40℃ and counterstained using the Mayer hematoxylin method, before being mounted and observed under light microscopy.