We also explored the fluorescence features of NRB-AF12 in intact tissues of dissected larvae of Drosophila melanogaster. Third instar larvae were labeled with NRB-AF12 and ER-Tr dissolved in the hemolymph-like HL3 saline, added to the medium following dissection and imaged with an immersion objective. As shown in Figure 9, a bright fluorescence was observed when larvae were treated with the fluorescent probes: In vivo live imaging of D. melanogaster muscle fibers (Figure 9A) and tracheal system (Figure 9B) revealed a sarcoplasmic reticulum distribution of the dyes, with NRB-AF12 and ER-Tr fluorescence significantly overlapping, as was observed in cell culture (Johnson et al., 2015). Finally, we investigated some physical properties of NRB-AF-12 such as penetration, internalization rate, retention time, washout time-course in living cells, as well as the induction of cytotoxicity, in order to establish the usefulness and reliability of the dye. Video 1 in Supplementary Material, shows that in living LX2 cells AF-12 is internalized in minutes and is retained in the cells for at least 3 h following its removal from the culture medium. Furthermore, re-exposure of the cells to dye, gave the same results, in terms of fluorescence distribution, compared to the first exposure. NRB-AF12 was apparently not toxic in both human and rat cell lines, even at concentrations much higher than those (500 nM) utilized for labeling (Figures 11B–D).