Tritc conjugated goat anti mouse antibody

Control 2/16 pairs of blastomeres and experimental embryo fragments (2/16 pairs and 4/32 quartets) were fixed in 4% PFA for 40 min at room temperature, and then permeabilized with 0.3% Triton X-100 for 15 min. The nonspecific antibody binding was blocked by incubation in 3% BSA (for CDX2 staining) or in 10% FBS (for p-Ezrin staining), overnight, at 4°C. Embryos and embryo fragments were then incubated with primary antibodies (mouse monoclonal anti-CDX2 antibody (1:50, BioGenex, USA) or rabbit monoclonal antibody anti-p-ERM (phospho-Ezrin(Thr567)/Radixin(Thr564)/Moesin(Thr558), 1:400, Cell Signaling Technology, 3141) at 4°C, overnight, and then treated with secondary antibodies (TRITC-conjugated goat anti-mouse antibody (1:200, Jackson ImmunoResearch) or goat anti-rabbit AlexaFluor 633 (1:200, Molecular Probes)) for 1 hour at room temperature. Additionally, chromatin was stained by incubation with DRAQ5 (10 μM; Biostatus, Leicestershire, UK) or chromomycin (0.01 mg/ml) for 10 min at 37°C, and F-actin by incubation with FITC-conjugated phalloidin (1:1000) for 20 min at room temperature. The embryos and embryos fragments were analyzed using a Zeiss 510 confocal laser microscope.