Mouse anti crb

Embryos were stained following standard protocols. Embryos were fixed in 4% formaldehyde (Sigma-Aldrich) in PBS1x-Heptane (1:1) for 10 min for Ecad staining and for 20 min for the rest. Embryos transferred to new tubes were washed in PBT-BSA blocking solution and shaken in a rotator device at room temperature. Embryos were incubated with the primary antibodies in PBT-BSA overnight at 4°C. Secondary antibodies diluted in PBT-BSA (and for the CBP staining) were added after washing and were incubated at room temperature for 2 h in the dark. Embryos were washed, mounted on microscope glass slides with Fluoromount-G (Southern Biotech) and covered with thin glass slides.
The following primary antibodies were used: rabbit anti-DSRF (1:500: produced by N. Martín in J. Casanova's lab); rat anti-Ecad (1:100; DCAD2, Developmental Studies Hybridoma Bank (DSHB)); goat anti-Ecad (1:600; Santa Cruz); mouse anti-Crb (1:10; Cq4, Developmental Studies Hybridoma Bank (DSHB)); mouse anti-Mega (1:50; kindly provided by Dr R. Schuh); goat anti-GFP (1:300; ABCAM); rabbit anti-GFP (1:300, Life Technologies); mouse anti-Sn (1:100; DSHB) and rat anti-Trh (1:100; produced by N. Martín in J. Casanova's lab). The following secondary antibodies were used: donkey anti-goat; anti-mouse; anti-rabbit; and anti-rat (1:300; Life Technologies).

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.