Distribution of Cy5.5 between blood and lymphatic vessels at 120 minutes after administration of Gd-Cy5.5. To confirm the results obtained in vivo, groups of mice (n = 3) were euthanized at 120 minutes after i.v. administration of Gd-Cy5.5 and the bladders were removed, frozen, and prepared for confocal analysis of the co-localization of Cy5.5 with CD31-positive vessels (green fluorescence in A and C) or LYVE-1-positive lymphatic vessels (green fluorescence in B and D). A and B are representative microphotographs of the same sub-epithelial region, whereas C and D are representative photomicrographs of the detrusor smooth muscle. Note that blood vessels are just underneath the urothelial cell layer (A), whereas lymphatic vessels are deeper in the submucosa (B). The white arrows in A indicate Cy5.5 (red) that is co-localized with LYVE-1-positive cells in B (yellow arrows). The cyan arrow in B indicates Cy5.5 (red) that is co-localized with CD31-positive blood vessels in A (orange arrows). In all figures, the red represents Cy5.5 accumulation and the blue stain is DAPI. The yellow fluorescence indicates co-localization of Cy5.5 with CD31-positive blood vessels (A) or LYVE-1-positive lymphatic vessels (B). Quantification of the fluorescent signals by image analysis is presented in Figure 9.