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. Author manuscript; available in PMC: 2009 Sep 1.
Published in final edited form as: Hear Res. 2005 Jun;204(1-2):156–169. doi: 10.1016/j.heares.2005.02.002

Figure 7. Immunocytochemical localization of GTTR and unconjugated gentamicin.

Figure 7

MDCK cells were treated with 5 μg/mL GTTR (A-F), or 300 μg/mL unconjugated gentamicin (G,H) for 2 hours, fixed with FA only, permeabilized with methanol, and immunolabeled. A) In cells incubated at 37°C, GTTR fluorescence occurs as both diffuse and punctate (arrowheads) cytoplasmic labeling, with labeled nucleoli (arrows, and inset) and trans-nuclear tubules (double arrowheads, inset). B) Gentamicin immunolabeling of GTTR seen in (A) also reveals diffuse cytoplasmic, and intracellular puncta (arrowheads) of labeling; with weak labeling of the nucleoplasm, and labeled trans-nuclear tubules (double arrowheads, inset). GTTR-labeled nucleoli are not immunolabeled (arrows). C) Merged images of (A) and (B), show co-localization of both GTTR and immunofluorescence as yellow in the cytoplasm, puncta (arrowheads), and intra-nuclear tubules (double arrowheads). D) Cells loaded with GTTR on ice reveal very few intracellular puncta of fluorescence (compared to A), and robust, diffuse cytoplasmic and weak nucleoplasmic labeling, with labeled trans-nuclear tubules (double arrowheads) and nucleoli (arrows, inset). E) Gentamicin immunolabeling (α-GT) of GTTR seen in (A) also reveals only diffuse cytoplasmic and nucleoplasmic labeling, with weakly labeled trans-nuclear tubules (double arrowheads, inset). GTTR-labeled nucleoli are not immunolabeled (arrows). F) Merged image of (D) and (E). G) Immunofluorescence of gentamicin-loaded cells incubated at 37°C ice reveals diffuse cytoplasmic and nucleoplasmic labeling, intracellular fluorescent puncta (arrowheads). Presumptive sites of nucleoli are not immunolabeled (arrows). H) Immunofluorescence of gentamicin-loaded cells incubated on ice reveals diffuse cytoplasmic and nucleoplasmic labeling, with few intracellular puncta of fluorescence. Presumptive GTTR-labeled intra-nuclear structures are not immunolabeled (arrows). I) Cells, without gentamicin or GTTR treatment, incubated on ice and immunoprocessed with primary and secondary IgG display negligible non-specific labeling. Scale bars = 10 μm.