Immunohistochemical Analysis of Tumor Samples

Orthotopic tumors were fixed in 10% formalin and embedded in paraffin. H&E staining and immunohistochemistry were performed using 5 µm thick sections [41] (link). The following antibodies were used: Ki67 (Novacastra Leica Microsystems, Buffalo Grove, IL), cleaved caspase 3 (Asp175, Cell Signaling Technology, Danvers, MA), and CD34 (MEC 14.7, Abcam, Cambridge, MA). Sections were incubated in HRP-labeled secondary antibody and staining was detected by DAB (Dako, Carpinteria, CA). TUNEL assay was performed using the In Situ Cell Death Detection POD Kit (Roche, Indianapolis, IN) according to the manufacturer’s protocol. Images were taken using an Olympus BX60 microscope (Olympus, Center Valley, PA) equipped with a QImaging EXI Blue camera and ImagePro software (Media Cybernetics, Atlanta, GA).