Immunohistochemistry of patient breast samples

Experiments involving patient breast samples were approved by West Virginia University Institutional Review Board. Formalin-fixed paraffin-embedded (FFPE) breast tumor tissue from patients was processed according to vendor’s manual instruction (Biocare) and following a verified protocol in the Pathology Laboratory of Translational Medicine at WVU. Briefly, 3 μm sections were deparaffinized on slides, quenched with hydrogen peroxide, and incubated in BK channel antibody (Sigma-Aldrich, HPA054648) Sigma at 4 °C for 4 min. Horseradish peroxidase-containing secondary antibody (UMap anti-RB, Roche, Diagnostic, Cupertino, CA) was then added for 8 min and developed using Biocare DAB (brown color). Hematoxylin was used as a counterstain (blue color).

Automated formalin-fixed, paraffin embedded immunohistochemical staining, to evaluate tumor antigen expression profiles, is available via the Ventana DISCOVERY automated IHC Platform. Breast tumor IHC slides stained with BK channel antibody were examined under an Olympus VS-120 slide scanner with a 10X Plan S Apo/0.40 NA objective, equipped with a color camera (Pike 505C VS50). The images were analyzed using OlyVIA (Olympus) and ImageJ (NIH). Percentage of area staining was used to quantify the protein expression levels in IHC slides.