Ventana discovery xt automated system

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.

Immunohistochemistry for RAD50 was performed on a 4um-thick FFPE tissue section of the tumor from the salvage nephrectomy (same specimen undergoing WGS). A primary monoclonal anti-RAD50 antibody was used diluted to 1:100, CC1 standard (ab89; Abcam, Cambridge, MA). Staining was assessed with a Ventana Discovery XT Automated System with DAB Map kit detection system (Ventana Medical Systems, Tucson, AZ).