Immunohistochemical Analysis of Breast Cancer

Formalin-fixed and paraffin-embedded tissue sections from invasive breast cancers were derived from the archive of the Department of Pathology, Seinäjoki Central Hospital. The study has been approved by the Scientific Committee of Seinäjoki Central Hospital, Finland. According to the Finnish national ethics committee regulations, informed consent was not considered necessary for this study. Immunohistochemical stainings of ER, PR, and Ki-67 tissue sections followed the recommended staining protocols [3 ]. The slides were stained using the BondMax staining robot (Leica Microsystems, Wetzlar, Germany). In brief, ER was detected using monoclonal antibody 6F11 (diluted 1:300, Leica Biosystems, Newcastle, UK), PR was detected using monoclonal antibody PgR636 (diluted 1:600, Leica Biosystems, Newcastle, UK), and Ki-67 was detected using monoclonal antibody MIB-1 (diluted 1:100, Dako, Carpinteria, CA, USA). Antigen retrieval was performed in Tris-EDTA buffer (pH 9, 100°C for 40 minutes). Bound antibodies were visualized using Bond Refine Detection kit (Leica Biosystems, Newcastle, UK). Immunoreaction was intensified using 0.5% copper sulfate (5 minutes). Hematoxylin counterstaining (1 minutes in ChemMate diluted 1:6, Dako, Carpinteria, CA, USA) was performed using PBS as bluing reagent. The samples were cleared with ethanol and xylene and mounted using standard procedures.