Rat monoclonal anti mouse cd31

The H22 tumor-bearing mice were injected with FITC-labeled CS NPs and PBA-CS NPs while the subcutaneously transplanted tumor volume reached an average size of about 200 mm³. These mice were sacrificed after 24 h injected and tumors were quickly excised. Tumors were washed with PBS (7.4) for three times and immersed in paraformaldehyde (4%) solution for 6 h at 4 ^oC. Afterwards, tumors were co-incubated with sucrose solution (25%) for overnight and frozen in O.C.T. embedding medium at -80 ^oC for 20 min. Tumors were then cut into 6 μm sections for further use. The sections were rehydrated in Triton X-100 solution (0.1% in PBS) for 15 min and incubated with BSA (3% in PBS) solution for 60 min at 37 ◦C. After then the sections were incubated with a primary monoclonal antibody (1:400, rat monoclonal anti-mouse CD31, BD Pharmingen, San Jose, California) for 60 min in a humidified chamber at 37 ◦C. Next, the sections were washed with tween 20 (0.05% in PBS v/v) for 5 min and then counterstained with an Alexa 594 conjugated donkey anti-rat secondary antibody (1:1000, Molecular Probes, Eugene, OR) and an Alex 488 conjugated streptavidin (1:1000; Invitrogen, Camarillo, CA) in a humidified chamber at 37 ◦C in the dark for 0.5 h. At last, the tumor sections were washed with PBS (7.4) for three times and stained with DAPI. The sections were then observed with a LSM 710.