Chromomap kit with anti rabbit omnimap

Xenografted tumors were fixed in 10% neutral buffered formalin, paraffin embedded, and hematoxylin-eosin-saffron (HES) stained. Outgrowths were analyzed by immunohistochemistry (IHC) for the expression of biomarkers. Immunostaining was performed on a Discovery XT Platform (Ventana Medical System, Tucson, AZ, part of Roche Diagnostics) with antigen retrieval using either EDTA buffer, pH 8.0 (CC1, Ventana Medical System) or citrate buffer 10 mM, pH 6.0 (CC2, Ventana Medical System). Primary antibodies were mostly monoclonal rabbit antibodies, and paired slides immunostained with rabbit IgG were used as negative controls. Incubation and color development involved anti-rabbit multimer secondary antibody (horseradish peroxidase complex) with DAB (3,30-diaminobenzidine tetrahydrochloride) as a substrate (ChromoMap Kit with Anti-rabbit OmniMap, Ventana Medical System). The IHC slides were scanned using a Pannoramic SCAN II (3DHISTECH). We then used the HALO software (Indica Labs) to quantify the expression levels of ER, pAkt (S473), and p-S6riboprotein (S235/6).

Paraffin-embedded breast tumors samples, obtained at the time of initial diagnosis, were retrieved from the archives of the Department of Biopathology at René Huguenin Hospital. Sections of 3 μm in thickness were cut with a microtome from the paraffin-embedded tissue blocks of normal breast tissue, pre-invasive lesions and IBCs (invasive breast cancer). Tissue sections were dewaxed and rehydrated through a series of xylene and ethanol washes. Immunostaining was performed on a Dako automated system. Primary antibody against PKD1 (Cell signaling, Danvers, MA) was incubated overnight at 4°C (dilution 1/100).
Patient-derived xenografts were fixed in 10% neutral buffered formalin and embedded in paraffin. Tissue sections were immunostained in a Discovery XT Platform (Ventana Medical System, Tucson, Arizona, USA, part of Roche Diagnostics) using EDTA buffer pH 8.0 (CC1, Ventana Medical System) for antigen retrieval. Primary antibody against PKD1 (Cell signaling, Danvers, MA) was incubated during 30 min at 37°C (dilution 1/100). After incubation with anti-rabbit secondary antibodies, slides were covered with the chromogenic substrate diaminobenzidine (ChromoMap Kit with Anti rabbit OmniMap, Ventana Medical System) and counterstained with hematoxylin.