The Use of Oocytes from Lutjanus Griseus as a Model to Compare the Immunofluorescence of Four Dyes: La Jolla Blue™, Cy5.5™, Fluorx™ and Fluorescein Isothiocyanate

Since our eventual goal is to distinguish with immunologic tools the early life history forms of specific species of the Lutjanidae, different fluorescent dye-antibody conjugates were investigated as a model for that utility. Immunofluorescent tests were carried out with two dyes that are excited in the near-infrared region of the spectrum, La Jolla Blue™ (LJB) and Cy 5.5™, and with two dyes excited at the lower part of the visible range, FluorX™ and fluorescein isothiocyanate (FITC). The LJB is a relatively new fluorescent probe that was produced from the phthalocyanine class of dyes. Each dye was covalently labeled to the IgG fraction of a goat antiserum produced to a highly purified 66 kDa glycoprotein found in soluble extracts of adults, juveniles, and oocytes of the gray snapper Lutjanus griseus. An epi-fluorescence microscope was used with a camera, image processor, color video monitor and printer, and a PC computer with software for image acquisition and light intensity measurement. First, we investigated the autofluorescence properties of the oocytes at 490 nm (low visible range) and 685 nm (near infrared). Second, because photobleaching is a phenomenon inherent in fluorescence probes, each immune IgG-fluorescent dye conjugate was incubated with the lutjanid oocytes for temporal assays at excitation wavelengths. Significant photobleaching was observed the first few minutes of excitation at 490 nm for oocytes reacted with the FluorX and FITC conjugates and for the Cy 5.5 conjugate at 685 nm. Oocytes were allowed to recover for 30 min, then re-excited at either 490 or 685 nm. The photobleaching effect was reversible only to a small degree. Little or no photobleaching occurred with La Jolla Blue during the 8 min excitation period at 685 nm, showing that the dye is superior when prolonged periods of specimen observation are necessary using fluorescence microscopy.