Fig. 4. Immunofluorescence localization of CrPRF and actin in Chlamydomonas vegetative cells (A-C,F) and mt+ gametes (D,E) reveals CrPRF localization in the cytoplasm and flagella. (A-C,E) Double labelled with anti-CrPRF (FITC) and anti-actin (Cy5) antibodies and imaged by confocal microscopy. (D-F) Single labelled and observed by wide-field fluorescence microscopy. CrPRF was relatively intensely stained throughout the cell body, including prominent staining in two CrPRF-enriched regions (A-E, arrowheads) located at the anterior end of the cells. These CrPRF-enriched regions were located at the base of the flagellae near the basal bodies and near the F-actin-containing fertilization tubule in mt+ gametes (E). The CrPRF-enriched spots were observed in all cells, irrespective of fixation condition, but staining was enhanced in cells first fixed by acetone (C). (Cells in A, B and E were first fixed in formaldehyde, cells in C and F were fixed in acetone and cells in D and F were fixed in paraformaldehyde followed by acetone.) Anti-CrPRF also stained flagellae. This is illustrated in (D) using wide-field fluorescence microscopy, and in comparison with the negative control image (F). Notably, flagellar staining was not illustrated in images A-C and E owing to the focal plane. For each image, cells selected were representative of the entire population. Scale bars, 5 µm.