Fluorescence Microscopy of Apoptosis Markers

DHE and H₂DCF-DA (both from Invitrogen/Molecular Probes) labeling of unfixed tissue was performed as described (Owusu-Ansah et al., 2008 (link)). TUNEL labeling (Roche) was done according to the manufacturer’s instructions. Antibody labelings were done on fixed tissue following standard procedures (Fan and Bergmann, 2014 (link); Fogarty and Bergmann, 2014 (link)). The following antibodies were used: cleaved caspase-3 (CC3; Cell Signaling Technology); NimC (kind gift of I. Andó) (Kurucz et al., 2007 (link)); MMP1 (Developmental Studies Hybridoma Bank (DSHB)) and pJNK (Promega). Secondary antibodies were donkey Fab fragments from Jackson Immunoresearch. Eye/antennal cephalic complexes were counterlabeled with the nuclear dye DAPI to visualize tissue outline. Images were taken with a Zeiss LSM700 confocal microscope. For quantification of confocal images, the ‘Record Measurement’ function of Photoshop was used. Clones were outlined and signal intensity determined. Multiple clones of five to ten imaginal discs per genotype obtained in three independent experiments were measured. Analysis and graph generation was done using GraphPad Prism 7.03. The statistical method and the P values are indicated in the figure legends.