Figure 13.

Co-localization of BCG-GFP with LRG47-positive vesicles in the mouse bladder urothelium. Figures 13 A-D are representative photomicrographs obtained 24 hours after bladder instillation of BCG-GFP (1.5 × 10⁷ CFU) into the bladder of anesthetized mice (see material and methods) and Figures 13 G-J are high magnifications of the same area depicted in Figures A-D. Figures 13 A and 13 G are DAPI [4',6-diamidino-2-phenylindole] that was used to highlight the nucleus. Figures 13B and 13H were stained with LRG-47. Figures 13C and 13I indicate the presence of BCG in the urothelium as indicated by the fluorescence of GFP in two major areas delimited by white circles. Figures 13D and 13J are merged photograph indicating that BCG-GFP is co-localized in vesicles positives for LRG-47 (yellow). White arrows in Figures 13 A-D point to GFP and rhodamine signals that are co-localized in the merge picture (Figure 13D). Next, pictures 13B and 13C were submitted to image analysis by the Interactive 3D Surface Plot [90], a plug-in for ImageJ software [23]. Figure 13E represents the Integrative 3D surface that translates the luminance of the images in Figure 13 B-C as height for the plot using the nearest neighbor sampling. For co-localization of BCG-GFP and LRG47-Rhodamine, pictures were converted to 8-bit gray scale images (fluorescence intensity range: 0 < 255) using Image J software [23] and the analysis was performed with the co-localization-finder plug-in [24]. The results presented in Figure 13F were electronically generated by the software resulting in a merged picture with white areas indicating the co-localization between BCG and LRG-47-positive vesicles and the Pearson's_correlation and overlap coefficient are provided. These results indicate that instillation of BCG-GFP into the mouse bladder, in the conditions described in this manuscript, does not result in urothelial injury and microscopic extravasation of BCG or cause artifacts, including forcing bacteria into the urothelium.