Avidin biotin horseradish peroxidase complex

Tissue sections (3 mm) were deparaffinized in xylene and rehydrated in an ethanol series. The sections were subsequently washed with ultrapure water and unmasked in citrate antigen unmasking solution (Mitsubishi Kagaku Iatron, Tokyo, Japan) in an autoclave for 10 min at 121 C. The sections were washed with ultrapure water and phosphate-buffered saline and then treated for 30 min with 0.03% hydrogen peroxide to block endogenous peroxidase activity. The sections were incubated with anti-MUC1 (1:100; Fitzgerald) for 60 min at 37 C. The bound primary antibody was detected by incubating an anti-mouse secondary antibody and avidin/ biotin/horseradish peroxidase complex (Dako Cytomation, Kyoto, Japan) with the sections for 10 min at room temperature. The labeled antigens were visualized by staining with the DAB kit (Dako Cytomation). Finally, the sections were counterstained with hematoxylin and examined under a microscope.
Analysis of immunohistochemically-stained slides was performed as previously described. 9 Cytoplasmic or membranous reactivity was classified as follows: 0 (no reactivity), 1 (reactivity in <10% of cancer cells), 2 (reactivity in >10% but <40% of cancer cells), or 3 (reactivity in >40% of cancer cells). For statistical analysis, classes 0 and 1 were defined as negative, and classes 2 and 3 were defined as positive.