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We describe a very efficient method for fluorescent labeling of acidic structures in denervated skeletal muscle with rhodamine B. Rhodamine B at 50 ng/ml gave selective and distinct segmental labeling of denervated muscle fibers after 5-min incubation at room temperature. Labeling was also achieved at 4 degrees C. The labeling was disrupted by the ionophores monensin and nigericin, suggesting a labeling confined to acidic structures. Rhodamine B co-localized with the lysosomotropic dye Lyso Tracker Green and a marker for endocytosis (fluorescein isothiocyanate-labeled dextran). Rhodamine B, which is highly lipophilic, showed pH-dependent fluorescence emission in saturated aqueous N-octanol. Tetramethylrhodamine showed similar characteristics for labeling of denervated muscle fibers and pH-dependent fluorescence in N-octanol. The carboxyl group present in these two compounds appears important, because structurally related compounds that either lack this group or have it esterified failed to label denervated muscle fibers and showed no pH-dependent fluorescence in N-octanol. The results suggest that rhodamine B labels acidic organelles belonging to the endosomal/lyosomal system of denervated skeletal muscle fibers. Nevertheless, it failed to label such organelles in a number of mammalian cell types other than denervated skeletal muscle fibers.

Rhodamine B, a fluorescent probe for acidic organelles in denervated skeletal muscle.