Lycopersicon esculentum

At thirteen weeks of age mice were perfused with 10 ml PBS followed by 10 ml of 2% paraformaldehyde (PFA). Sixteen mice (8 per group) received retro-orbital injection of 150 μl FITC-lectin solution (0.5 mg/ml; Lycopersicon Esculentum, Vector Laboratories) prior to perfusion. Mice were anesthetized with 3% avertin by intraperitoneal injection prior to retro-orbital injection and perfusion. Lung and breast tissue were removed and mammary tumors dissected and weighed. Tumors and lung were cryopreserved in 30% sucrose at 4°C overnight. The FITC signal was enhanced with an anti-FITC antibody (71-1900; Life Technologies, Carlsbad, CA, USA; 1:500).
Six to eight images per tumor were taken at random with a Nikon Eclipse 90i microscope (with a DS-Qi1Mc monochrome CCD camera) (Nikon Instruments Inc., Melville, NY, USA) and NIS Elements AR 3.2 software (Nikon Instruments Inc.) using a 20X objective (Plan Apo 0.75; Nikon Instruments Inc.). The proportion of perfused vessels was counted per 20X field.

Tumor‐bearing mice were injected intravenously with 0.05 mg fluorescein isothiocyanate (FITC)‐conjugated lectin (Lycopersicon esculentum; Vector Laboratories) and perfused (lectin⁺) tumor vessels were counted on tumor sections. Tumor‐bearing mice were injected i.p. with 60 mg/kg pimonidazole hydrochloride and tumor hypoxic (pimonidazole⁺) areas were evaluated on paraffin sections immunostained with the Hypoxyprobe‐1‐Mab1 (Hypoxyprobe kit, Chemicon) following the manufacturer's instructions.

To visualize blood vessels, SCID mice were first anesthetized and given intracardiac injections of 100 μg DyLight 594 labeled tomato lectin (Lycopersicon esculentum; Vector Laboratories, Clinisciences). After 5 min, the heart was perfused with PBS-BSA 1% and with 4% paraformaldehyde. Lungs were then frozen and 60 μm-thick sections cut. The slides were mounted with Fluoromount G (SouthernBiotech), and cells counterstained with DAPI to localize the nucleus. A laser-scanning microscope (Zeiss LSM 700) in the tile scan mode was used to capture a mosaic of images.

To detect endothelial cells, 20-μm sections were incubated with 5 ug/mL FITC-labeled tomato lectin (Lycopersicon esculentum) (Vector Laboratories, Inc.), which recognizes endothelial cell walls, for 4 h at 4°C. Cellular nuclei were stained with DAPI. The fluorescence images were observed by a Keyence microscope. The volume of FITC labeling in the penumbra and ischemic core was computed using the image analysis software supplied with the microscope (Keyence, Osaka, Japan). Total FITC signal intensity was counted in 3 sections of penumbra area/mouse.

For lectin perfusion, the mice were i.v. injected with 100 μg of FITC‐labelled tomato lectin (Lycopersicon esculentum; Vector). After 10 min of circulation, mice were heart‐perfused with 2% neutral‐buffered formalin (w/v) and tumours were frozen in O.C.T. compound. To evaluate tumour hypoxia, mice were i.v. injected with 1.5 mg pimonidazole (Hydroxyprobe™‐1). Tissues were collected after 1‐h circulation, fixed with 2% neutral‐buffered formalin and frozen in O.C.T. after 2 h in 10 and 30% sucrose gradients.