Making walled-, highly autofluorescent dinoflagellate algae cells accessible and amenable for immunofluorescence and application of fluorescent probes

Raúl E. Castillo-Medina, Georgina Arzápalo-Castañeda, Marco A. Villanueva

Limnol. Oceanogr. Methods 9:460-465 (2011) | DOI: 10.4319/lom.2011.9.460

ABSTRACT: Immunofluorescence or fluorescent probe procedures with dinoflagellate algae have been hampered by their intrinsic pigment autofluorescence and rigid cell walls that cannot be digested by cellulolytic or pectinolytic enzymes. Highly autofluorescent, chlorophyll-containing cells from Symbiodinium were fixed and treated by a freeze-fracture procedure on microscope slides, intended to preserve their structure, break the cell wall, eliminate endogenous fluorescent pigments, and provide accessibility to antibodies. Formaldehyde fixation was carried out on intact cells in suspension, followed by freeze-fracture in liquid nitrogen. Finally, they were subjected through a series of tests for pigment extraction to eliminate the cell autofluorescence. Incubation in 70% isopropanol, followed by NaBH4 after freeze-fracture on glass slides, proved to be the most effective treatment to remove the fluorescence from endogenous pigments and free aldehyde groups remaining after the fixation. The procedure was effective in preserving the cell structures during the lengthy incubations, and the cells were suitable for both, immunolocalization of internal antigens, and visualization of targets of fluorescent probes. The procedure was equally successful when applied to other Symbiodinium species. These results provide a powerful tool for performing immunofluorescence and fluorescent probe detection on these highly autofluorescent, chlorophyll-containing cells.