Most current approaches to multiantigen fluorescent imaging require overlaying of multiple images taken with separate filter sets as a result of differing dye excitation requirements. labeled secondary antibodies were then used to simultaneously mark four antigens in fixed cells, using a single image and filter set. We also imaged different surface tumor markers on two live cell lines. Experiments showed that all colors could be visualized simultaneously by eye under the microscope, yielding multicolor images of multiple cellular antigens in real time. for details). The fluorescent chromophore was assembled in five steps from benzothiadiazole (Scheme?1) via known intermediate 4. It was brominated and then coupled to Calcitetrol a known deoxyribose precursor via Heck coupling and converted to the deoxyriboside (7) in two standard steps. Two additional routine reactions were carried out to provide the 5-dimethoxytrityl-3-phosphoramidite derivative (9) for use on a DNA synthesizer. Absorption and emission spectra of the deoxyriboside are given in Fig.?S1; the compound has long-wavelength absorption bands at 330 and 490?nm and a prominent emission peak at 670?nm. Scheme 1. Reagents and conditions: (a) Br2, 48% HBr, 72%; (b) 1,3,5-Trioxane, 48% HBr, AcOH, Trimethyl(tetradecyl)ammonium bromide, 51%; (c) NaIO4, dimethylformamide, 90%; (d) 4-(Dimethylamino)phenylboronic acid, Pd(Ph3P)4, Na2CO3, toluene, 92%; (e) 2-Amino-5-bromobenzenethiol, Calcitetrol … Fig. 1. Structures in this study. (670?nm. These ODFs as antibody conjugates were compared in a fixed-cell assay (see Fig.?S2 and for details). The emission spectra of ODF-conjugated antibodies (Fig.?2were adjusted simultaneously. Multiplex Labeling of Nucleoli, Nuclei, Mitochondria, and Microtubules. Fixed and blocked HeLa CCL-2 cells were incubated with 5?g/mL mouse anti-OxPhos complex V inhibitor protein (Invitrogen) mixed with prediluted human antinuclear antigen antibody (Inova Diagnostics) at room temperature for 1?h, followed by incubation in 10?g/mL 5-SSEYK-labeled goat anti-mouse IgG (Invitrogen) mixed with 10?g/mL 5-SSYZY labeled goat anti-human IgG (Thermo Scientific) at room temperature for 1?h. Then the cells were rinsed and incubated sequentially with 1 blocking buffer at room temperature for 1?h, 5?g/mL rat anti–tubulin at room temperature for 1?h, and 10?g/mL 5-SSEY labeled goat anti-rat IgG at room temperature for 1?h. Finally, the cells were rinsed and incubated sequentially with 1 blocking buffer at room temperature for 1?h, 5?g/mL mouse antinucleolin antibody (Invitrogen) at room temperature for 1?h, and 10?g/mL 5-SB labeled goat anti-mouse IgG at room temperature for 1?h. Stained cells were mounted on slides in Cytoseal 60 (Thermo Scientific) with coverslips. Samples were imaged under a Nikon Eclipse E800 epifluorescence microscope equipped with 100 objective. Images were Rabbit Polyclonal to HDAC5 (phospho-Ser259). captured using a Spot RT digital camera Calcitetrol and Spot Advanced Imaging software with excitation 340C380? nm and emission >?420?nm. Contrast and brightness of all the real-color images shown in Fig.?4 were adjusted simultaneously. Detection of CEA on Tumor Cell Surfaces. Cultured HeLa CCL-2 cells, LS 174T cells, and the mixed two cell lines were rinsed with PBS and were subsequently incubated with 5?g/mL of 5-SSEY conjugated goat anti-human CEA antibodies (BiosPacific) at 4?C for 1?h. Then the cells were rinsed with PBS again before imaging, which was carried out using a Zeiss LSM 510 confocal laser scanning microscope through a 40 objective. The argon laser (458?nm) was used to excite 5-SSEY (Fig.?S9). Image acquisition was performed at the Cell Sciences Imaging Facility of Beckman Center for Molecular and Genetic Medicine, Stanford University Medical Center, Stanford, CA. Detection and Differentiation of Live Tumor Cells. Cultured A431 cells (ATCC), LS 174T cells (ATCC), and the mixed two cell lines were rinsed with PBS and were subsequently incubated with 5?g/mL of 5-SSYZY conjugated goat anti-integrin antibodies (Santa Cruz Biotechnology) and 5?g/mL of 5-SSEY conjugated goat anti- human CEA antibodies (BiosPacific) at 4?C for 1?h. Then the cells were rinsed with PBS again before imaging, which was carried out using a Nikon Eclipse E800 epifluorescence microscope through a 20 objective. Images were captured using a Spot RT digital camera and Spot Advanced Imaging software with excitation 340C380?nm and emission >?420?nm..