Ab214275

To begin with, 3 μm sections from the FFPE of 14 CRC patients’ biopsies were immunohistochemically stained using the rabbit monoclonal antibody for HER2 (1:4000 dilution; ab214275, Abcam, Waltham, MA, USA) according to the manufacturer’s instructions. An experienced pathologist (R.H.) scored the stained slides, following the consensus recommendations for HER2 scoring for CRC [17 (link),18 (link)]. Briefly, scoring was performed on a 4-point scale—0, 1+, 2+, 3+ focusing on intensity and extent according to the Allred scoring system [19 (link)]. In this study, 0 and 1+ intensity were taken to be negative for HER2 expression and the study focused on the assessment of membranous HER2 expression.

All IHC staining of tumor xenografts was performed by the Duke Research Immunohistology Laboratory on a fee-for-service basis. Harvested tumor tissue was fixed in 2% neutral buffered formalin and embedded in paraffin. Thin, ~5 µm tissue slices were cut using a microtome, and the sections were mounted for antigen retrieval. Standard immunohistochemistry methods of deparaffinizing and rehydrating with solvents were used, followed by immunostaining of tissue sections with primary antibody against HER2 (Abcam cat# ab214275) at 1:100 dilution for 45 min at room temperature and then labeling with HRP-conjugated secondary detection antibody for 30 min at room temperature and detected with Vector’s RTU Elite ABC reagent (Cat# PK-7100) for 30 min at room temperature. Underlying tissue structures were visualized by counterstaining with Harris hematoxylin.

Anti-HER2 (ab214275, Abcam, Cambridge, UK), actin (A2066, Sigma-Aldrich, Oslo, Norway), goat-anti-rabbit-HRP (65–6120, ThermoFisher, Oslo, Norway), goat anti-mouse-AF488 (ab150117, Abcam, Cambridge, UK), goat anti-rat-PE (405406, Biolegend, Oslo, Norway), goat anti-rat-AF546 (A11081, ThermoFisher, Oslo, Norway), goat anti-rabbit-AF647 (A27040, ThermoFisher, Oslo, Norway), horse-anti-mouse-HRP (7076S, Cell Signaling Technology, Danvers, MA, USA) and Fixable Viability Dye eFluor™ 780 (eBioscience, Oslo, Norway).