Figure 1C shows phase contrast microscopy images and conventional epifluorescence microscopy images of living LX2 cells incubated with 500 ηM NRB-AF12. The images clearly show a meshwork distribution of the fluorescence with a sharp demarcation of the nuclei and an accumulation in perinuclear areas; moreover, NRB-AF12 did not stain either the nuclei or the plasma membrane. NRB-AF12 internalization into the cell was fast, commencing after only a few seconds and reaching completion within a few minutes, following its addition to the incubation medium. A similar fluorescence pattern was also observed in fixed LX2 cells (Figure 1D), however, the NRB-AF12 signal was not retained post-permeabilization (data not shown). The sub-cellular distribution of NRB-AF12 was investigated in more detail in LX2 cells using confocal microscopy in combination with fluorescent probes and/or antibodies targeted to specific subcellular structures. To evaluate the distribution of NRB-AF12 in the ER, LX2 cells were transfected with the ER-reporter red fluorescent protein (RFP)-KDEL plasmid and subsequently loaded with the dye. The results, reported in Figures 2A,D, show that the NRB-AF12 signal significantly overlapped with that of (RFP)-KDEL (Pearson's Coefficient: 0.21 ± 0.10; n ≥ 10), but a larger staining distribution of NRB-AF12 was observed which indicated other targets in addition to ER. Similar results were observed in LX2 fixed cells labeled with NRB-AF12 and immunostained for the ER resident protein Calreticulin (Pearson's Coefficient: 0.3 ± 0.06; n ≥ 10) (Figures 2B–D). We also compared the staining pattern of NRB-AF12 with that of ER-Tr, a commercial fluorescent probe widely used to label ER. Surprisingly, co-labeling of LX2 cells with NRB-AF12 and ER-Tr resulted in a strong overlap between fluorescent dyes (Figure 2C), confirmed by the high rate of co-localization index (Pearson's coefficient: 0.75 ± 0.02; n ≥ 15). This remarkable similarity between the two dyes was further supported by the observation that ER-Tr, as well as NRB-AF12, were not selective for the ER, as demonstrated by co-staining LX2 cells with ER-Tr plus either RFP-KDEL (Figure 3A) or Calreticulin (Figure 3B) (Pearson's coefficient: 0.23 ± 0.1 and 0.29 ± 0.03, respectively for RFP-KDEL and Calreticulin; n ≥ 10; Figure 3C).