Ventana discovery xt autostainer platform

For immunohistochemical analysis of MDAMB453 xenografts, tumours were extracted 4 hours post-dose and formalin fixed for immunohistochemistry (IHC). Each sampling time point includes 4 animals /treatment group. IHC was performed on 4um thick formalin-fixed, paraffin-embedded tissue sections mounted on glass slides. For cleaved caspase 3 (Cell Signaling Technologies, Danvers, MA), staining was performed on a DAKO autostainer. Sections were treated with DAKO Target Retrieval (Dako; Carpinteria, CA), incubated with primary antibody at 0.12ug/ml overnight at 4°C followed by biotinylated goat anti-rabbit IgG (Vectorlabs, Burlingame, CA) and detected with Vectastain ABC-HRP (Vectorlabs, Burlingame, CA). For phospho-S6, IHC was performed on the Ventana Discovery XT autostainer platform (Ventana Medical Systems Inc, Tucson, AZ). The slides were pre-treated with CC1, standard time, followed by anti-phospho-S6 (Cell Signaling Technologies, Danvers, MA), incubated at 0.26ug/ml for 32 minutes at 37°C. The antibody was detected with anti-rabbit-UltraMap (Ventana Medical Systems Inc, Tucson, AZ). Staining was visualized with DAB. Sections were counter stained with hematoxylin, dehydrated, cleared and cover-slipped for viewing.