Rat anti mouse cd31 monoclonal antibody

We defined pericyte-covered vessels as endothelial cells were fully surrounded or partially surrounded by αSMA positive cells as described previously [33 (link),34 (link)]. Briefly, 8-μm frozen sections were fixed with cold acetone and immunostained with rat anti-mouse CD31 monoclonal antibody (BD Biosciences, Franklin Lakes, NJ) and mouse anti-α-SMA monoclonal antibody (Sigma-Aldrich) by the ABC and LSAB methods using a Vectastain ABC kit (Funakoshi, Tokyo, Japan) and LSAB kit (Dako, Glostrup, Denmark). Adjacent sections were routinely stained with hematoxylin and eosin. All histological specimens were viewed under a CCD Hyper Scope (Keyence, Osaka, Japan) and analyzed using Image Tool software (Image Tool, Roswell, GA). MVD was determined as the mean of four or five fields per cross-section. Pericyte coverage was calculated based on the ratio of the double-staining vessels per total vessel count in the hotspot areas. Human tumor specimens were purchased from SuperBioChips (Seoul, South Korea). The human tumor specimens were double-stained with mouse anti-human CD31 monoclonal antibody (Dako) and alkaline phosphatase-labeled mouse anti-α-SMA monoclonal antibody (Sigma-Aldrich) using an Envision HRP kit (Dako) and the LSAB kit.

Ethanol, fibrinogen, aprotinin and thrombin were purchased from Sigma-Aldrich (St. Louis, MO, USA), and SU5416 was purchased from Calbiochem (San Diego, CA, USA). Rat anti-mouse CD31 monoclonal antibody was obtained from BD Biosciences (San Diego, CA, US), while anti-VEGF antibody was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Cytodex 3 beads were obtained from Amersham Pharmacia Biotech Inc. (Piscataway, NJ, USA).

Eight micrometer-thick frozen tumor sections were washed in phosphate-buffered saline (PBS) and blocked in 5% normal donkey serum in an antibody dilution buffer consisting of PBS containing 0.1% Triton X-100. Sections were then incubated overnight in primary antibody (rat anti-mouse CD31 monoclonal antibody; Clone: MEC 13.3, 1:200 dilution, BD PharMingen, San Diego, CA) or rabbit anti-mouse LYVE1 polyclonal antibody (1:200 dilution, Angiobio Co., Del Mar, CA) at 4 °C, and labeled with a fluorescein-conjugated secondary antibody (Molecular Probes, Leiden, The Netherlands). Nuclei were counterstained with DAPI and are seen in blue. Samples were observed with a fluorescence microscope (Olympus, Tokyo, Japan). The number of lymphatic vessels within the tumor was counted in six fields per section: the center regions of the tumor (to measure intra-tumor vessel density) and within an area 1 mm from the tumor border (to measure peri-tumor vessel density). Seven slide sections per mouse were analyzed. To determine the metastasis of SW620 tumor cells to the mouse lateral axillary lymph nodes, lungs, and liver, sections of mouse lymph nodes, lungs, and liver (seven slide sections per mouse) were stained with H&E and rabbit anti-CCR7 monoclonal antibody (Clone: Y59, 1:200 dilution, Abcam Inc. Cambridge, MA).

Surgically removed Panc02 tumors were embedded and frozen in OCT compound. Cryostat sections (8-μm thick) were fixed in 4% paraformaldehyde for 10 min and blocked with 1% BSA in DPBS. The sections were incubated with rat anti-mouse monoclonal CD31 antibody (1:100 dilution) (BD Biosciences, Franklin Lake, NJ, USA) followed by incubation with Alexa Fluor 594-conjugated goat anti-rat IgG (1:300 dilution) (Thermo Fisher Scientific) and counterstaining with DAPI. Images were taken with a confocal laser scanning microscope (TCS SC8, Leica Microsystems, Wetzlar, Germany), and pixel values of the CD31-positive areas were calculated for each image to determine the tumor vessel density using ImageJ software (National Institutes of Health).

Vessel density in P29mtP29 and P29mtB82M tumours was determined by staining tissue sections for CD31. For this purpose, surgically removed tumours were immediately embedded and frozen in optimal cutting temperature (OCT) compound. Next, cryostat sections (8-µm thick) were cut and fixed in 4% paraformaldehyde for 10 min, blocked with 1% bovine serum albumin (BSA) in Dulbecco’s phosphate-buffered saline (DPBS) and then incubated with rat anti-mouse monoclonal CD31 antibody (BD Biosciences, 550274, 1:100). After being washed with DPBS, the sections were incubated with Alexa Fluor 594-conjugated goat anti-rat IgG (Invitrogen, Thermo Fisher Scientific, A-11007, 1:300) for 1 h. After counterstaining with 4,6-diamidino-2-phenylindole (DAPI), the sections were observed under a confocal laser scanning microscope (Fluoview FV1000, Olympus, Tokyo, Japan). The pixel values of the CD31-positive areas were calculated for each image to determine the tumour vessel density using the ImageJ software (National Institutes of Health).