Vectra automated quantitative pathology imaging system

Sections were stained using the Bond RX autostainer (Leica Biosystems Inc., Buffalo Grove, IL, USA). Briefly, slides were baked at 65 °C for 15 min and the automated system performed dewaxing, rehydration, antigen retrieval, blocking, primary antibody incubation, post primary antibody incubation, detection (DAB), and counterstaining using Bond reagents (Leica). Samples were then removed from the machine, dehydrated through a series of ethanol and xylenes and mounted. Rabbit anti-γ-H2AX (Ser139) (1:800, #9718, Cell Signaling), rabbit anti-phospho-EGFR (Tyr1068) (1:50, #2234, Cell Signaling), rabbit anti-phospho-HER2/ErbB2 (Tyr1221/22) (1:400, #2243, Cell Signaling), rabbit anti-PTEN (1:150, #9559, Cell Signaling: used for mouse transplants and normal reduction mammoplasty samples), mouse anti-PTEN (1:100, #M3627, Dako: used for HER2 breast cancer patient cohort), rabbit anti-Ki67 (1:200, #ab16667, Abcam) were diluted in antibody diluent (Leica). TUNEL staining was performed using manufacturer’s recommendations (ApopTag Peroxidase In Situ Apoptosis Detection Kit, #S7100; EMD Millipore). All microscopic imaging was done using the VECTRA^® Automated Quantitative Pathology Imaging system or an Axioskop 40 with ZEN Software (Zeiss, Germany). Whole tissue imaging was done using a Stemi SV 11 Stereoscope with ZEN Software (Zeiss).