Immunofluorescence Analysis of PAR Protein Distribution

For immunofluorescence analysis, embryos were fixed in methanol and stained as described previously [29 (link)]. Images were acquired using a Zeiss LSM 510 confocal microscope. The primary antibodies used were rabbit anti-PAR-6 (1/50, [15 (link)]) and mouse P4A1 anti-PAR-3 (1/150, Developmental Studies Hybridoma Bank). Secondary antibodies were Alexa488-coupled goat-anti-rabbit and Alexa546-coupled goat-anti-mouse (1/500 each, Invitrogen). The cortical distribution of PAR-3 and PAR-6 proteins was measured using Image J software by plotting fluorescence intensity values of a 10 pixel-thick line drawn around the entire cortex of the embryo. This produced a fluorescence intensity profile where a broad peak of intensity corresponding to the entire anterior cortex is bordered by regions of low/background intensity in the posterior pole. The two points on each side of the broad peak where fluorescence intensity starts to rise were considered as PAR protein cortical boundaries and were determined as the intersection between the average fluorescence intensity of the posterior cortex and that of the slopes on each side of the peak. PAR protein domain size in each embryo was expressed as the distance between the anterior pole and the average of the two intersecting points relative to embryo length.