Immunostaining of Drosophila Embryos

Embryos were fixed in two different ways. For staining with anti-Crumbs (Cq4) and anti-Armadillo (N27A1) antibodies (both from the Developmental Studies Hybridoma Bank), we fixed the dechorionated embryos by a two second heat treatment at 93.4 °C, basically as described in Miller, Field & Alberts (1989) (link). For the anti-Coracle (C566.9) and anti-Fasciclin 3 (7G10) stainings (antibodies also from the Developmental Studies Hybridoma Bank), we fixed dechorionated embryos according to Krahn et al. (2010) (link). The rest of the protocol was according to Miller, Field & Alberts (1989) (link). Anti-Crumbs was used at a 1:20 dilution, anti-Coracle at a1:200, anti-Fasciclin 3 at 1:200, and anti-Armadillo at a1:20. Secondary antibody was anti-mouse conjugated with Cy-3 (Invitrogen) at 1:1,000. For nuclei, we used Sytox-Green (1:3,000) according to the manufacturer’s instructions (Invitrogen). Embryos were mounted in Vectashield (Vector Laboratories), and imaged using a Zeiss 780 confocal microscope with 25× and 63× objectives. Images were acquired with Zeiss software, and manipulated using ImageJ (NIH). We used stage 13–14 embryos for the Crumbs, Coracle, and Armadillo antibody stainings; for the Fasciclin 3 staining, we used stages 15–16 embryos, except for Fig. S1, where stages 13–14 embryos were used. Finally, in Figs. 1C and 2 stage 16 embryos were used.