Lasersharp 2000 software

The FLP/FRT system [61 (link)] was applied for generating twin clones as described [43 (link),62 (link),63 (link)] by crossing the FRT82B H^ΔCT fly line with y¹w* hs-flp; FRT82B Ubi-GFP^S65Tnls/TM6B (BL32655, obtained from BDSC, Bloomington, IN, USA); the wild-type allele hence expressed GFP. Clones were induced by a 1 h heat shock at 37 °C in first to second instar larvae. Wing imaginal discs were dissected from wandering third instar and subjected to antibody staining according to standard procedures [62 (link),63 (link)] using primary rat anti-Deadpan antibodies (Dpn, 1:100; ab19573 Abcam, Cambridge, UK) and goat secondary antibodies coupled to Cy3 (Jackson Immuno-Research via Dianova, Hamburg, Germany). GFP signals were recorded without further enhancement. Fluorescently labeled tissue was embedded in Vectashield (Vector labs, Eching, Germany) and pictures taken with a BioRad MRC1024 confocal microscope coupled to a Zeiss Axioskop using LaserSharp 2000^TM software (Carl Zeiss, Jena, Germany). The figures were assembled using Image J, Corel Photo Paint and Corel Draw software.

Immuno-staining of imaginal discs was according to standard protocols using Hairless anti-A (1:500, from guinea pig) [21 (link)], mouse anti-Cut (1:25) or anti-Wingless (1:25) (developed by G. Rubin and S.M. Cohen, respectively; obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Dept of Biology, Iowa City, IA 52242) and rabbit anti-GFP (1:200, Santa Cruz Biotech, Dallas, USA). Goat secondary antibodies coupled to FITC, Cy3 or Cy5 were from Jackson Immuno-Research (Dianova, Hamburg, Germany). Tissue was mounted in Vectashield (Vector Labs, Eching, Germany), and examined using a BioRad MRC1024 confocal microscope and LaserSharp 2000TM software (Carl Zeiss, Jena, Germany).