Ventana omnimap anti mouse secondary antibody

Immunostaining for ERBB4 was performed using a Ventana Discovery XT Automated System (Ventana Medical Systems, Tucson, AZ). Briefly, slides were deparaffinized on the automated system with a preparatory solution. A heat-induced antigen retrieval method was used in cell conditioning 1. A mouse monoclonal antibody that reacts to ERBB4, NB100–2662 (Novus, Littleton, CO), was used at a 1:25 concentration in Antibody Diluent (Dako, Carpenteria, CA) and incubated for 60 minutes. The Ventana OmniMap Anti-Mouse Secondary Antibody (Ventana Medical Systems) was incubated for 16 minutes and detected using a ChromoMap kit (Ventana Medical Systems). Slides were then counterstained with hematoxylin and eosin.

All Cali samples were shipped as formalin-fixed paraffin-embedded (FFPE) tissue blocks to Moffitt Cancer Center, Tampa, FL for immunohistochemical analysis, as previously described (31 (link)). All samples from both cohorts were stained using the Ventana automated immunostainer Discovery XT (Ventana, Tucson, AZ, USA) and Ventana ChromoMap kit (Ventana). Tissue sections of 4 μm were incubated for 2 h with mouse anti-human CD133 monoclonal antibody (1:100; MAB4399, Millipore, Billerica, MA, USA), followed by incubation with the Ventana OmniMap anti-mouse secondary antibody (Ventana) for 16 min. Kidney tissue samples were used as the positive control, while ommission of the first antibody served as negative control. Slides were counterstained with H&E. All sections were dehydrated and coverslipped as per normal laboratory protocol. Slides were read by two pathologists. The immunostain for CD133 was localized to the cytoplasm in all cases. At each disease stage, extent of immunohistochemical staining (of epithelial cells) in terms of both percentage staining and staining intensity was evaluated. The Allred scoring system featuring a proportion score (scale of 0–5) and an intensity score (scale of 0–3) was utilized to give a total positivity score between 0 and 8 (32 (link)).