RHODAMINE-B, A FLUORESCENT-PROBE FOR ACIDIC ORGANELLES IN DENERVATED SKELETAL-MUSCLE

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 denerv ated muscle fibers after 5-min incubation at room temperature, Labelin g was also achieved at 4 degrees C, The labeling was disrupted by the ionophores monensin and nigericin, suggesting a labeling confined to a cidic structures, Rhodamine B co-localized with the lysosomotropic dye Lyse Tracker Green and a marker for endocytosis (fluorescein isothioc yanate-labeled dextran), Rhodamine B, which is highly lipophilic, show ed pH-dependent fluorescence emission in saturated aqueous N-octanol. Tetramethylrhodamine showed similar characteristics for labeling of de nervated muscle fibers and pH-dependent fluorescence in N-octanol. The carboxyl group present in these two compounds appears important, beca use 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 rhoda mine B labels acidic organelles belonging to the endosomal/lysosomal s ystem 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.