AN EMPIRICAL PROTOCOL FOR WHOLE‐CELL IMMUNOFLUORESCENCE OF MARINE PHYTOPLANKTON 1

An immunofluorescence protocol for intracellular antigens of phytoplankton was developed empirically. Paraformaldehyde (4%) was used to fix samples for 6 h at 4° C; fixed samples can be stored in cold (–20°C) methanol for at least 1 month. We used Triton X‐100, Nonidet P‐40, and dimethyl sulfoxide to permeabilize the cells for antibody penetration across the cell wall and plasma membrane. After immunolabeling at room temperature, the samples were mounted on slides and could be stored at 4° C for up to 6 months without significant decay of the fluorescence. The staining was examined with an epifluorescence microscope; semiquantitative (percentage of cells with positive staining) and quantitative (staining intensity measured with an imaging system) analyses were performed. Reproducible staining was achieved for nuclear, chloroplastic, cytoplasmic, and cell surface antigens in species of Chlorophyceae, Prymnesiophyceae, Bacillariophyceae, and Dinophyceae including some field‐collected samples. This protocol is advantageous to previously published protocols in that it allows relatively simple and long‐term storage of fixed samples and allows staining of multiple antigens for the same sample. Although improvement on cell permeabilization is required for some species, the present protocol could prove useful for physiological or ecological studies when frequent sampling is required and immediate processing of the samples is not possible.