Mouse anti 2a12

Drosophila embryos were formaldehyde-fixed and processed as described previously (Tepass et al., 1990 (link)). For Arm staining, embryos were heat-fixed (Gamblin et al., 2014 (link)). Primary antibodies used: mouse anti-2A12 (1:10 dilution; Developmental Studies Hybridoma Bank, DSHB), mouse anti-Cora (1:500; clones C566.9 and C615.16, DHSB), mouse and anti-Tango (1:10; DHSB), mouse anti-Arm (1:250; N2-7A1, DHSB), mouse anti-Fas3 (1:10; 7G10, DHSB), rabbit anti-Verm (1:500; Luschnig et al., 2006 (link)), rabbit anti-Serp (1:500; Luschnig et al., 2006 (link)), rabbit anti-aPKC (1:250; C-20, Santa Cruz Biotechnology) and rat anti-Crb (1:500; this study). Secondary antibodies were conjugated to Cy3 (Jackson Immunoresearch Laboratories) or Alexa Fluor 488 (Molecular Probes). Rhodamine-conjugated Chitin-Binding Probe (New England BioLabs) was used at a concentration of 4 µg/ml, and co-incubated with secondary antibodies.

We followed standard protocols for immunostainings and in situ hybridisations. Embryos were staged as described [42 ]. Imaginal discs were obtained by dissecting third instar larvae.
The following primary antibodies and dilutions were used: mouse anti-2A12 (recognises Gasp, 1:10), rat anti-DEcad (1:100), and mouse anti-Crb (1:20) from Developmental Studies Hybridoma Bank, DSHB; rbb anti-Verm (1:300) from S. Luschnig; goat anti-GFP (1:600) Molecular Probes and Roche; ck anti-ßGal (1:500) abCAM; GP anti-Uif (1:400) from R. Ward; and rbb anti-Pio (1:100) from M. Affolter. CBP (chitin-binding probe) conjugated with Cy3, Cy2 and Cy5 was used at 1:300 (generated by N. Martin). WGA conjugated with Alexa-555, -488, and -647 was used at 1:300 (Molecular Probes). Cy3-, Cy2- and Cy5-conjugated secondary antibodies (Jackson ImmunoResearch) were used at 1:300.
A reb riboprobe was generated using the following primers:

Forward: 5′- AACTGTGCCTCGGCGCTAGTC

Reverse: 5′- AGCAGTCGAAACACGCAGCTT

Confocal images were acquired with a Leica TCS-SPE system. Images were post-processed with ImageJ and Adobe Photoshop and assembled using Adobe Illustrator.