Figure 1: Distinct profiles of monoclonal antibodies recognizing IZUMO1 in cell–oocyte assay.

(a) Immunostaining and cell–oocyte assay of a COS-7 cell expressing Izumo1 and DEL5-113. COS-7 cells that were transfected with mouse IZUMO1 and the truncated DEL5-113 cDNA were stained with Mab17-Alexa546 (red), Mab18-Alexa488 (green) and Mab125-Alexa647 (magenta) simultaneously after dissociation by 10 mM EDTA containing PBS, as described in Methods. The final concentration of all antibodies added was at 0.5 μg ml⁻¹. Scale bars, 20 μm. Right photographs show the transmission images of the cell–oocyte assay. Scale bars, 100 μm. (b) Cell–oocyte assay with IZUMO1-specific antibodies. Izumo1-expressing COS-7 cells and oocytes were incubated for 2 h at 37 °C with the monoclonal antibodies in a (at the same concentrations). Inset shows differential interference contrast (DIC) image of the centre of the Z-stacks. Some attached COS-7 cells are out of focus. The adherent surface of COS-7 cells to the oocyte is indicated by asterisks. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm. The outline of the oocyte is marked by dotted lines. (c) The relative fluorescence intensities at the interface. The × 2 magnified image of the white dashed box in b is shown. The relative fluorescence intensity in the white arrow line of the dashed box in c was analysed with ImageJ. The fluorescence intensities of Mab17-Alexa546, Mab18-Alexa488 and Mab125-Alexa647 are indicated in red, green and purple lines, respectively. The arrow indicates a measurement direction.