Alexafluor488 labeled goat anti human antibody

Tumors were extracted, and sectioning was performed by the University of Wisconsin Carbone Cancer Center Experimental Pathology Laboratory. Frozen tumor tissue slices of 5-mm thickness were fixed with cold acetone for 10 min, washed with PBS, and then blocked with 10% donkey serum for 45 min at room temperature. Slices were then incubated overnight with pertuzumab (10 μg/mL) and rat anti-mouse CD31 (2 μg/mL; ThermoFisher Scientific) at 4 ^°C. Next, sections were further stained with AlexaFluor488-labeled goat anti-human antibody and Cy3-labeled donkey anti-rat antibodies (ThermoFisher Scientific). A coverglass was applied to each slide using Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and imaging was performed using an A1R confocal microscope (Nikon, Nikon Instruments, Melville, NY, USA).