Mouse β galactosidase β gal antiserum

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).

Embryo fixation and antibody staining were performed as previously described [7 (link)]. The following antisera were used at the indicated dilutions: rat dCREB antiserum at 1:10 000; rat antisera to DE-cadherin at 1 : 400 (Developmental Studies Hybridoma Bank, DSHB; Iowa City, IA); rabbit aPKC antiserum (Santa Cruz Biotechnology, Dallas, TX) at 1 : 500; mouse β-galactosidase (β-gal) antiserum (Promega, Madison, WI) at 1 : 500. Appropriate AlexaFluor 488-, 647- or rhodamine- (Molecular Probes, Eugene, OR) conjugated secondary antibodies were used at a dilution of 1 : 500 for salivary glands. Anti-mouse-Cy5 secondary antibody was used at 1 : 200 dilution for wing discs (Jackson, West Grove, PA). 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 an LSM 710 for laser scanning confocal microscopy.
To collect wing discs, the larvae were dissected in 1× phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 30 min at room temperature, washed with 1× PBS for 10 min, followed by a 5 min wash with PBTx (0.1% Triton X-100). The wing discs were stained with 10 µg ml⁻¹ Hoechst33342 in PBTx for 2 min, washed three times in PBTx and mounted on glass slides in Fluromount G (SouthernBiotech).