Wing imaginal disc immunofluorescence

The anterior halves of third instar larvae of third instar, 5-7-day old, homozygous mutant for the indicated genes, larvae were dissected in phosphate-buffered saline (PBS), everted, and fixed in 4% formaldehyde at room temperature for 45 min. They were washed three times in PBS+0.1% Triton-X (PBSTx), blocked in 5% normal goat serum for one hour at room temperature, and incubated overnight at 4°C in a 1:100 dilution of cleaved Dcp-1 antibody (Cell signaling #9578S) in PBSTx. Larva heads were then washed six times with PBSTx and incubated in a 1:500 dilution of secondary antibody (goat anti-Rabbit IgG, Alexa Fluor 488, Life Technologies) for two hours at room temperature. After washing six times in PBSTx, DAPI was added at a 1:1000 dilution. Discs were dissected and mounted in 50 ul of Fluoromount G mounting media (Thermo).

Pictures were taken with a Zeiss LSM880 confocal laser scanning microscope using a 40X oil immersion objective with a constant gain and a 0.6X zoom using ZEN software. Images were saved as.czi files and were processed and the signal was quantified using ImageJ as in [54].