Immunohistochemical staining was performed using standard procedures (see Supplemental Methods ). Rac1 was stained with mAb (clone 102, BD Biosciences, 10155-1-AP) and Cdc42 was stained with rabbit pAb (Protein Tech Group). A Vectastain Ready-to-Use (RTU) ABC-peroxidase kit and ImmPact DAB (SK-4105) were used to visualize primary antibody labeling with hematoxylin nuclear counterstain (H-3401) for tissue staining and samples were mounted in VectaMount (H-5000); (all from Vector Laboratories).
For large scale ovarian tumor profiling, tissue microarrays were purchased from US BioMax, Inc. (Rockville, MD, cat# OV1005 061 and OV8010 009). In total 180 unique tissue samples were included in the evaluation; ranging from stage I-IV and grades 1–3 (seeSupplemental Table S1 ). All tumor types were validated and staining was scored by a pathologist with gynecologic pathology specialty (Dr. Lomo) and evaluated for location (nuclear and cytoplasmic), as well as intensity of positive staining. Scoring was based on the product of the percentage of cells stained and the intensity of the staining in each localization (3+: strong, 2+: intermediate, 1+: weak and 0: no staining), resulting in a minimum of 0 (100% cells×0) and a maximum of 300 (100% cells × 3+).
For large scale ovarian tumor profiling, tissue microarrays were purchased from US BioMax, Inc. (Rockville, MD, cat# OV1005 061 and OV8010 009). In total 180 unique tissue samples were included in the evaluation; ranging from stage I-IV and grades 1–3 (see