(A) Micrographs showing fluorescent labeling of cell using NAI-A594 and M1-A488. Images in (a) demonstrate that following labeling of receptors with NAI-A594, the treatment with naloxone (1 µM, 10 min, n = 3) did not reduce the fluorescence along the plasma membrane. Images in (b) show the fluorescence of A594 from the reversible ligand NTX-A594 labeling was weaker and abolished following treatment with naloxone (1 µM, 10 min, n = 3). In both (a) and (b), the fluorescence of antibody labeling using M1-A488 wasere not changed after the treatment of naloxone. (B) Flow cytometry shows an increase in fluorescence with increasing concentrations of NAI-A594 and NTX-A594. The red line shows the signal after wash with a buffer [2 × 2 mL DMEM (no phenol red) plus 5% FBS, room temperature]. The red dashed-line shows fluorescent signal after wash with naloxone (10 µM, 2 × 2 mL, room temperature, n = 3). The black lines (both solid and dashed, n = 3) represent cells treated with NTX-A594 and washed as described for NAI-A594 treated cells. When cells were co-incubated with naloxone (10 µM), there was negligible NTX-A594 fluorescence (black dot). The fluorescence of antibody labeling M1-A594 was also quantified as shown with a blue square. (C) Immunoprecipitation-western blot analyses show the labeling of FMOR with NAI-A488 (100 nM, 1 hr, 37°C, 5% CO2). The solubilized receptors from these cells were immunoprecipitated with rabbit anti-MOR and then immunoblotted with primary rabbit anti-Alexa488 and secondary goat-anti rabbit Alexa-680 (lanes 1–4, left panels) or primary M1 anti-FLAG and secondary goat-anti mouse Alexa-680 (lanes 1–4, right panels). lane 1: HEK293 cells that lack FMOR, lane 2: unlabeled FMOR cells, lane 3: FMOR cells labeled with NAI-A488 in the presence of 10 µM naloxone, and lane 4: NAI-A488 labeled FMOR cells. The experiments were repeated twice. (D) The fluorescent spectra of solubilized receptors in (C) were measured between wavelengths of 450–600 nm. Only the receptors that were incubated with NAI-A488 alone showed significant fluorescence. The fluorescence was blocked by 10 µM naloxone, was at a low level from unlabeled FMOR cells and was not observed in HEK cells without FMOR. (E) The labeling of purified FMOR by NAI-A488 increased with time. FMOR was purified and incubated in NAI-A488 at room temperature for the indicated time. Band intensities were measured using image J. The error bars represented standard deviation of two independent experiments. (F) Micrographs of cells co-labeled with NAI-A594 and M1-A488. Images show the plasma membrane staining of M1-A488 (a) and NAI-A594 (b) and the appearances of fluorescent puncta in M1-A488 (c) and NAI-A594 (d) labeled cells after application of 10 µM ME for 10 min.