Localization of TGF-β1 in rat bladder. Control and CYP-treated bladders were harvested at 24 h after CYP injection, fixed in formalin, and cryopreserved prior to sectioning to a thickness of 8 μm. Bladder sections were stained for TGF-β1 (red fluorescence) and LAP (blue fluorescence) for the immunodetection of active and latent TGF-β1, respectively. The urothelium region of sections was marked by a lower degree of green stain for smooth muscle actin/phalloidin. Male CYP-treated rats exhibited the most intense magenta stain to indicate the substantial presence of active TGF-β1 in urothelium (Panel D; lumen marked by white arrow), that was much lower in control male rats (Panel C) and nearly absent in female control rats (Panel A). The purple color emerging from the predominance of blue fluorescence in the overlap with red fluorescence was more prominent in controls of both genders as well as in female rats treated with CYP, but absent in male CYP-treated rats. Magnification is 60× in all sections and is representative of 4 animals in each group. The experiment is representative of 5 fields per slide.