Live young adult animals were immobilized with 0.1% tricaine/0.01% tetramisole and mounted on 2% agarose pads at 20°C. Alae defects phenotypes were observed by differential interference contrast using a Leica DMRXA2 6000 equipped with a Coolsnap HQ (Roper Scientific) camera with a 100× objective (oil; NA 1.4). To follow the localization of fluorescent proteins, Z-stacks (0.3-µm spacing) were acquired using a DMI6000 (Leica) spinning disk (Yokogawa CSU22 with an Andor iXonEM+ 897 camera) with a 63× objective (oil; NA 1.4). Leica Type F immersion medium was used. Young adults were observed (right after alae formation and before formation of the first embryos). Identical settings were used for control and mutant animals. Image analysis was performed using ImageJ software (National Institutes of Health). Colocalization (Fig. 1, Fig. 2, and Fig. 4) was quantified using a semiautomated method (comparison of local intensity maxima obtained in each channel) and a manual method based on scan line analysis (comparison of fluorescence intensity profile along a line crossing a fluorescent punctum in a given channel with the profile obtained from the same line in the other channel), which was previously described (Hyenne et al., 2012 (link)).