Embryo Fixation and Antibody Staining

Embryo fixation and antibody staining were performed as previously described [21 (link)]. The following antisera were used at the indicated dilutions: rabbit Vermiform antiserum at 1:300 (a gift of S. Luschnig); rat Dysfusion antiserum at 1:200 (a gift of S. Crews); mouse 2A12 antiserum at 1:10 and rat E-cadherin antiserum at 1:20 (Developmental Studies Hybridoma Bank, DSHB; Iowa City, IA); guinea pig Arl3 antiserum at 1:200 (a gift of L. Jiang); mouse DSRF antiserum at 1:100 (Active Motif, Carlsbad, CA) and mouse β-galactosidase (β-gal) antiserum at 1:500 (Promega, Madison, WI). Appropriate biotinylated- (Jackson Immunoresearch Laboratories, Westgrove, PA), AlexaFluor 488-, 647- or Rhodamine- (Molecular Probes-Thermofisher Scientific, Waltham, MA) conjugated secondary antibodies were used at a dilution of 1:500. F-actin was detected with phalloidin (1:20; Invitrogen-Thermofisher Scientific) as previously described [22 (link)]. Stained embryos were mounted in Aqua Polymount (Polysciences, Inc., Warrington, PA) and thick (1 μm) fluorescence images were acquired on a Zeiss Axioplan microscope (Carl Zeiss) equipped with LSM 510 for laser scanning confocal microscopy at the Weill Cornell Medical College optical core facility (New York, NY).