Reduced colocalization of fibrinogen degradation products (FDPs) and CD31 in Src-deficient mice. (A) Indirect immunofluorescence of cryosections of control (src^+/–) and Src-deficient (src^–/–) primary D121 subcutaneous tumors (12 day) immunostained with an antifibrinogen antibody (labeled with a green secondary antibody, left column) and an anti-CD31 antibody (labeled with a red secondary antibody, middle column). Images are merged in the right-hand column. Quantitation of the degree of colocalization was performed on multiple representative tumor cryosections immunostained to detect CD31 and fibrinogen. The Pearson coefficient for colocalization of CD31 and fibrinogen in control D121 tumors (src^+/– and wild type) was 0.72+/–0.04 and for src^–/– mice it was 0.37 +/– 0.11 (n = 6 for each, P < .004). These coefficients were calculated as described in “Materials and methods.” Bar = 50 μm. (B) Micrograph of hematoxylin/eosin-stained primary D121 tumors grown in src^+/– and src^–/– mice at low (top row) and high (bottom row) magnification. Micrographs are representative of triplicate staining assays. Bar = 33 μm. (C) Tumor-induced VP was determined in D121 tumor-bearing lungs from src^+/– and src^–/– by intravenous injection of 70-kDa FITC-dextran, removal of the intact lungs, homogenization, and quantitation with a fluorescent spectrophotometer as described in “Materials and methods.” (*P < .05; n = 4). Error bars indicate standard error. (D) Indirect fluorescent immunostaining with anti-VEGF (labeled with a green secondary, top row) and anti-CD31 (labeled with a red secondary, bottom row) antibodies of cryosections of primary D121 tumors grown in src^+/– and src^–/– mice (n = 3). Bar = 100 μm. (E) Whole tissue lysates were prepared from D121 primary tumors (top row) and secondary spontaneous lung tumors (12 day, bottom row) from src^+/– and src^–/– mice. Immunoblots are representative of triplicate Western blots.