Goat anti rat cy5

The eye imaginal discs were dissected from the third-instar larvae in S2 insect medium. Apoptosis was determined by staining with 3 mg/ml of acridine orange (Sigma-Aldrich). For cell proliferation, dissected discs were labeled with BrdU (bromodeoxyruidine, Becton Dickson) as described [39 (link)]. Briefly, BrdU labeled eye discs fixed in PBS/4% paraformaldehyde (PFA), were denatured by HCl, and neutralized by PBS. Apoptosis was analyzed with a Zeiss fluorescent microscope. In order to generate gain-of-function clones, the FLP-out GAL4 system (flipase driven GAL4 expression) was employed [40 (link)]. In brief, virgin females hsflp;act>y⁺>GAL4UASGFP/CyO were crossed with w+;UAS-DRhoGEF2/UAS-DRhoGEF2 or w+;UAS-mycPDZ-RhoGEF/UAS-mycPDZ-RhoGEF at 18°C for 3 days, then, parental flies were flipped out. Embryos were heat shocked for 45 min at 37°C and maintained at 25°C. Eye imaginal discs from wandering third-instar larvae were dissected and fixed in PBS/4% PFA (Sigma-Aldrich), washed in PBS/0.1% Triton X-100 (Sigma-Aldrich), and incubated overnight with primary antibody. Discs were stained with rat anti-Elav (Developmental Studies Hybridoma Bank, University of Iowa), goat-anti-rat-Cy5 (Jackson Lab), and phalloidin-rhodamine (Molecular Probe). The stained discs were analyzed with a Zeiss confocal microscope.