Rabbit anti fak antibody

Preimplantation mouse embryos were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at 4°C, followed by a washing in PBS containing 1% BSA (PBS-BSA) three times. The embryos were then permeabilized by a 5-min incubation in PBS containing 0.1% Tween 20 at room temperature. After blocking for 30 min at room temperature in 10% normal non-immune rabbit or goat serum in PBS, the embryos were incubated with one of the following primary antibodies at 1∶100 dilution: rabbit anti-neogenin antibody (Santa Cruz Biotech, Santa Cruz, CA), goat anti-DCC antibody (Santa Cruz Biotech), rabbit anti-FAK antibody (Santa Cruz Biotech), and rabbit anti-integrin β1 antibody (Santa Cruz Biotech) in 1% PBS–BSA overnight at 4°C. After three washes with PBS, the embryos were incubated with either Alexa 488-conjugated goat secondary IgG or Alexa 568-conjugated goat secondary IgG (Invitrogen, Grand Island, NY) at 1∶500 dilution overnight at 4°C. Visualizing actin filaments were made by incubating embryos in 1 µg/ml phalloidin for 1 hr at room temperature. The nuclei were stained with DAPI at 1 µg/ml for 5 min. After three more washes with PBS, the embryos were transferred into a PBS drop on a glass slide. Fluorescent images of the stained embryos were acquired with LSM700 META confocal microscope (Carl Zeiss AG, Oberkochen, Germany).