Alexa 546 coupled goat anti mouse

Hippocampal neuronal cultures on Poly-Ornithine-Laminin coated coverslips were fixed for 15′ at RT in 3.7% formaldehyde. After three washes in PBS, fixed cells were permeabilized using 0.5% Triton-X-100 for 5 min and afterwards incubated in PBS containing 2% goat serum and 0.1% Triton-X-100 for 60 min to block nonspecific binding. Cultures were incubated for 2 h with primary antibodies in the presence of goat serum (2%) and 0.1% Triton-X-100. The primary antibodies used were anti-Syt-1 (1∶1000, mouse monoclonal, Synaptic Systems, Germany, Cat.No. 105 011), and anti-Syt-7 (1∶1000, rabbit polyclonal, Abcam, USA, Cat.No. 105 173). The cells were washed three times with PBS and then incubated over night at 4°C with secondary antibodies: Alexa 546-coupled goat-anti-mouse (Invitrogen) and Alexa 637-coupled goat-anti-chicken (Invitrogen), both diluted 1∶500. Immunofluorescence images were taken with a confocal microscope (LSM 510 utilizing 488 nm, 543 nm and 633 nm lasers controlled by LSM 5 software attached to an Axiovert 200; Zeiss, Germany) using a 63× oil immersion (1.4 NA) objective. For the 546 nm channel, a HFT 488/543 Dichroic Beam Splitter (BS) and a 560–615 BP Filter were used; HFT 488 BS and 505–530 nm BP Filer for the 488 nm channel and a HFT UV/488/543/633 combined with a 650 nm LP filter for the 633 nm channel (all Zeiss, Germany).

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.