Four weeks after MBT-2 cell implantation, bladder and SLN were sampled. For whole-mount staining, tissue samples were fixed in 1% paraformadehyde in PBS for 1 hr at room temperature (RT). Whole-mounted tissues were incubated for 3 hr at RT with hamster monoclonal anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) antibody 1:1,000 (Chemicon International, Temecula, CA), or rabbit polyclonal anti-lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) antibody 1:1000 (Upstate, Lake Placid, NY) after a blocking the fixed tissues with 5% donkey serum in PBST (0.3% Triton X-100 in PBS) for 1 hr at RT. Samples were incubated for 2 hrs at RT with HRP-conjugated anti-hamster antibody 1:100 (Jackson ImmunoResearch, West Grove, PA) or HRP-conjugated anti-rabbit antibody, 1:100 (Amersham, Piscataway, NJ) and developed with 3,3'-diaminobenzidine (DAB) substrate kit according to manufacturer's instruction (Vector, Burlingame, CA). Samples were analyzed and photographed with a Zeiss Stemi SV6 stereomicroscope. For hematoxylin-eosin (H&E) and immunofluorescent staining of tissue sections, Samples were fixed in acetone for overnight at -20°C. Samples were embedded in paraffin block after serial incubation with methyl benzoate and xylene for 30 min at RT. Paraffin blocks were cut in 5-10 μm sections using Microm HM 335E rotary microtome (IMEB Inc, San Marcos, CA). Tissue sections were blocked with 5% goat or donkey serum in TBST (0.03% Triton X-100 in TBS) after dissolving the paraffin with xylene and acetone, immunostained with antibodies for 3 hr; these included hamster monoclonal anti-PECAM-1 antibody 1:200, rabbit polyclonal anti-LYVE-1 antibody 1:200, rat monoclonal anti-CD11b antibody 1:100 (BD Pharmingen, Franklin Lakes, NJ), rat monoclonal FITC-conjugated anti-CD11b antibody 1:50 (BD Pharminogen), mouse monoclonal anti-Cytokeratin antibody (Abcam Inc, Cambridge, MA), rat monoclonal FITC-conjugated anti-CD68 antibody 1:100 (Serotec Ltd., Oxford, UK), rat monoclonal anti-F4/80 antibody 1:100 (Research Diagnostics Inc., Flanders, NJ), goat polyclonal anti-VEGFR-3 antibody 1:100 (R&D Systems, Inc., Minneapolis, MN), goat polyclonal anti-VEGF-C antibody 1:100 (Santa Cruz Biotechnology, Inc.). After several washing steps using TBS, FITC-conjugated anti-hamster IgG antibody 1:1000, FITC-conjugated anti-rat IgG antibody 1:1000, FITC-conjugated anti-mouse IgG antibody 1:1000, Cy3-conjugated anti-rabbit IgG antibody 1:1000, Cy3-conjugated anti-rat IgG antibody 1:1000, Rhodamine-conjugated anti-goat IgG antibody 1:1000 were used as secondary antibodies. All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. Goat Fab fragment anti-mIgG (Jackson ImmunoResearch Laboratories, Inc.) was used to block endogenous mouse IgG to use mouse antibody on mouse tissue. Fluorescent signals were visualized and digital images were obtained using a Zeiss LSM 510 confocal microscope equipped with argon and helium-neon lasers (Carl Zeiss, Germany) or using a Zeiss ApoTome microscope coupled to a monochrome charge-coupled device camera (AxioVision, Carl Zeiss). Morphometric measurements of lymphatic and blood vessels in tumor and SLN were made from immunostained tissue sections by photographic analysis using ImageJ software (
http://rsb.info.nih.gov/ij) after converting the images into 8-bit gray scale. Measurements of the number and size of the lymphatic vessels having lumen in tumor section were made at a screen magnification of 100×, each 0.84 mm
2 in area. Measurements of the densities of the blood vessels in tumor section were made at a screen magnification of 100×, each 0.84 mm
2 in area, whereas those and pan-cytokeratin stained cancer cells in SLN were made in total sectioned area (0.3-1.2 mm
2), and 4 to 5 mice were used per group. To exclude background fluorescence, only pixels over a certain level (>50 intensity value) were taken. Measurements of the densities of the lymphatic vessels and blood vessels in the whole mount staining of bladder were made on whole fields at a screen magnification of 25×, relative densities were calculated in each 1.16 mm
2 in area, and 4-5 mice were used per group. Values were expressed as relative densities (%). To measure the size of tumor and SLN, the mid-sectioned tissues were stained with H&E. The stained tissues were photographed with a Zeiss microscope, surface areas of mid-sectioned tumor and SLN were calculated using a Zeiss Apotome microscope coupled to monochrome charge-coupled device (CCD) camera and image analysis software (AxioVision, Zeiss).