Alexa488 coupled goat anti rabbit

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.

Thoracic mammary glands were fixed for 24 hours in 4% paraformaldehyde and embedded in paraffin wax. Paraffin sections of 5 µm were prepared and subjected to 1 mM disodium-EDTA antigen retrieval as described previously [28] (link). Primary antibodies used for immunofluorescence are the following: Cytokeratin 8 (Developmental Studies Hybridoma Bank TROMA-I, rat, 1∶100), Estrogen receptor (Novocastra NCL-ER-6F11, mouse, 1∶100), E-Cadherin (BD Biosciences 610181, mouse, 1∶250), Progesterone receptor (Abnova MAB9785, rabbit, 1∶400), Smooth muscle actin (Sigma A2547, mouse, 1∶1000), Tbx3 (Invitrogen 424800, rabbit, 1∶100), turboGFP (Pierce Antibodies PA522688, rabbit, 1∶400), turboGFP (OriGene TA150041, mouse 1∶250). Secondary antibodies used at 1∶400 dilution are from Invitrogen: Alexa488-coupled goat anti-mouse (A11029), Alexa488-coupled goat anti-rabbit (A11034), Alexa568-coupled goat anti-mouse (A11031) and Alexa568-coupled goat anti-rabbit (A11036). Additionally, CF633nm-coupled donkey anti-rat (Biotium 20137-1) was used at 1∶400 dilution. Images were acquired on a Zeiss LSM-710 confocal microscope with a pinhole aperture of 1 Airy unit.