Rat anti e cadherin

Fixed embryos were analyzed in Figs. 7, S1 B, S2 (C and D), and S3 (D and E). All other images shown in this study are of living embryos. To visualize α-catenin and armadillo/β-catenin, embryos were boiled for 10 s in 0.03% Triton X-100/0.4% NaCl and devitellinized in heptane/methanol. To visualize Myo-GFP and E-cadherin, embryos were fixed for 10 min in a 1:1 mixture of 37% FA/0.1 M phosphate buffer, pH 7.2, and hand devitellinized in 0.1 M phosphate buffer. Antibodies used were anti–α-catenin (1:50; Developmental Studies Hybridoma Bank), rabbit anti–armadillo/β-catenin (1:100, made by J.A. Zallen as described by Riggleman et al., 1990 (link)), rat anti–E-cadherin (1:50; Developmental Studies Hybridoma Bank), and rabbit anti-GFP (1:150; Torrey Pines). Secondary antibodies conjugated to Alexa Fluor 488, 546, or 647 fluorophores (Molecular Probes) were used at a concentration of 1:500. Embryos were mounted in ProLong Gold (Invitrogen) between two coverslips for imaging. Images were acquired on a Zeiss LSM 700 confocal microscope with a PlanNeo 40×/1.3-NA oil-immersion objective (1.1-µm optical section and 0.56-µm z-steps).

Embryos were dechorionated in 50% bleach for 2 min and washed with water. For live-imaging experiments, dechorionated embryos were mounted between a coverslip and a gas-permeable membrane (YSI, Yellow Springs, OH) in halocarbon 27 oil (Sigma). For immunofluorescence, embryos were fixed with vigorous shaking in a 1:1 mix of 37% formaldehyde and heptane for 7 min, and manually devitellinized. Embryos were stained in 1X phosphate-buffered saline or 0.1 M Na₂HPO₄ (pH 7.2) (for E-cadherin antibodies) and mounted in Prolong Gold (Molecular Probes). Primary antibodies were rat anti-E-cadherin (1:50, Developmental Studies Hybridoma Bank [DSHB]), rabbit anti-GFP (1:100, Torrey Pines Biolabs), mouse anti–α-tubulin (1:500, DSHB), and rabbit anti-mud (1:50, gift of F. Matsuzaki, RIKEN Center for Developmental Biology, Kobe, Japan; Izumi et al., 2006 (link)), and were detected with Alexa Fluor 488-, 546-, 568-, and 647-conjugated secondary antibodies (1:500, Molecular Probes). F-actin was visualized with Alexa Fluor 488-, 546-, 568-, or 647-conjugated phalloidin (1:1000, Molecular Probes) or Alexa Fluor 405-conjugated phalloidin (1:1000, Santa Cruz). Guinea pig anti-Bazooka (Par-3) (1:500) (Blankenship et al., 2006 (link)) was included with phalloidin in the red channel in Figure 7B but is not visible at the gain shown. Analysis of denticle cells was conducted on abdominal segments 3–7.