Rat anti mouse pecam cd31

The following antibodies were used for tissue immunohistochemistry: rat anti-mouse PECAM/CD31 (BD Biosciences 553370); rabbit anti-GLUT1 (Thermo Fisher Scientific RB-9052-P1). Alexa Fluor-labeled secondary antibodies and GS Lectin (Isolectin GS-IB4) were from Thermo Fisher Scientific.
The following antibodies were used for immunoblot and immuno-staining analysis: mouse anti-1D4 (MacKenzie et al., 1984 (link)); rabbit anti-6xMyc (JH6204); rabbit anti-Reck (Cell Signaling 3433); rat anti-alpha tubulin (Invitrogen MA1-80017); and AP-conjugated horse anti-mouse IgG antibody (Vector Laboratories AP-2000). Fluorescent secondary antibodies for immunoblotting were from Li-Cor.

Mouse tumor tissues were fixed in 10% buffered formalin solution (Sigma-Aldrich) at room temperature and infiltrated with 20% sucrose overnight at 4 °C. Tumor tissue blocks were then imbedded with OCT Tissue-Tek compound (SAKURA, West Chester, PA, USA) onto a PVC plastic mold and rapidly frozen to −30 °C in dry ice, 15 μm thick sections (at least 10 slices per block) were obtained using a Leica CM1850 cryostat (Leica Microsystems, Wetzlar, Germany). Frozen sections were dried for 2 h at room temperature, washed with PBS three times for 3 min, and surface tensions reduced using an application of 0.05% triton X-100 2 times for 5 min. Sections were blocked in 5% normal goat serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 h, incubated overnight in a humidified chamber with each primary antibody, 1:200 Rat-anti mouse PECAM (CD31) and Rat-anti mouse VE-Cad (CD144) (Both from BD bioscience, La Jolla, CA, USA), washed with PBS with 0.1% Triton X-100, and then incubated for 1 h with secondary antibodies 1:500 FITC goat anti-rat IgG (BD bioscience) at room temperature. After washing with PBS, samples were mounted with ProLong Gold anti-fade regent containing 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen), and confocal fluorescence images were acquired using the FV10i FLUOVIEW Confocal Microscope (Olympus, Tokyo, Japan).