Publication protocol
Tissue sections (6 μm) were stained using a standard protocol. Briefly, slides were heated for antigen retrieval by pressure cooker treatment in 0.01 M sodium citrate buffer, pH 6.0 (125°C for 3 minutes, 90°C for 10 seconds). Sections were blocked in 3% (v/v) normal goat serum in 0.05% (v/v) PBS-Tween 20 for 1 hour at room temperature. Primary antibody incubation was conducted in blocking buffer overnight at 4°C. Non-specific rabbit IgG (Dako, Campbellfield, Victoria, Australia) or mouse IgG (Dako) antibodies were used as isotype controls. Biotin-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Dako) were used at 1:250 or 1:300 dilution for 1 hour at room temperature. Specific primary-secondary antibody complexes were detected using ABC reagent (Vector Laboratories, Burlingame, CA, USA) and visualised using a 3, 3′-diaminobenzidine peroxidase substrate kit (Vector Laboratories). Sections were counterstained with hematoxylin, dehydrated and mounted. The primary antibodies and dilutions used were as follows: mouse monoclonal anti-human ERα (1:100, clone 1D5, Dako), chicken polyclonal anti-human ERβ (1:500, a kind gift from Dr Jan-Åke Gustafsson, University of Houston, TX, USA), rabbit polyclonal anti-human PR (1:4500, sc-538, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-human HER2 (1:400, A0485, Dako), mouse monoclonal anti-human cytokeratin 5/6 (KRT5/6, 1:100, clone D5/16 B4, Merck Millipore, Kilsyth, Victoria, Australia), rabbit polyclonal anti-EGFR (1:50, ab2430, Abcam, Cambridge, UK) and rabbit polyclonal anti-mouse p53 (1:300, CM5p, Novocastra).
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