Mouse anti nrt

Embryos were methanol-heat-fixed (Miller et al. 1989 (link)) and stained with rabbit anti-Zip (1:200), rabbit anti-Anillin (1:100) (Goldbach et al. 2010 (link)), mouse anti-Nrt (1:10, Developmental Studies Hybridoma Bank, DSHB), and mouse anti-Arm-N27A1 (1:50, DSHB). Embryos were fixed in 4% formaldehyde/phosphate buffer with heptane (Karr and Alberts 1986 (link)), and stained with mouse anti-Dlg (1:20; DSHB), and mouse anti-Pnut (1:10; DSHB). Goat anti-mouse and anti-rabbit secondary antibodies were conjugated to Alexa Fluor 488, 546, or 680 (Invitrogen). Embryos were fixed in 8% formaldehyde/phosphate buffer with heptane (Warn and Magrath 1983 (link)), and stained with Alexa Fluor 488-conjugated phalloidin (Invitrogen) to visualize F-actin. Embryos were mounted in Aquapolymount (Polysciences, Warrington, Pennsylvania), and imaged using an Olympus FluoView 300 or a Nikon Ti-E A1 confocal microscope. Image analyses were performed using ImageJ (Schneider et al. 2012 (link)). Circularity index (c = 4πA/p², where A = area, p = perimeter) was determined (Thomas and Wieschaus 2004 (link)), and tested using a two-sided Mann-Whitney test. Circularity index data from wild type and drak^del were compared to circularity index data from drak^del lines carrying phosphomimetic sqh transgenes, and nonphosphorylatable sqh transgenes, using a Kruskal-Wallis test with Dunn’s post-test.

Following documentation, the cover slip was cut into small pieces each carrying a single embryo and set on small applejuice-agar petri-dishes, provided with yeast. Hatching larvae were grown until late L3, when their CNS was prepared as described elsewhere (Bello et al., 2007 (link); Shimosako et al., 2014 (link)). For fibre tract staining we used mouse-anti-Nrt (1:10) (Hortsch et al., 1990 (link)) (Developmental Studies Hybridoma Bank), which was incubated for several days at 4°C and anti-mouse-Alexa647 (1:500) (Life Technologies) as secondary antibody. Specimens were embedded in Vectashield Mounting Medium (Vector Laboratories) and documented as described above, but scanned at 600 Hz with two frame averages and an additional channel for the Nrt-staining. Most times 70–80 slices were recorded with a thickness between 1–1.5 µm.

Drosophila melanogaster embryos were fixed using the heat/methanol protocol [62 (link)]. Fixed embryos were stained with mouse anti-Nrt (1:10, Developmental Studies Hybridoma Bank, DSHB), rabbit anti-Zip [63 (link)] and Hoechst dye. Secondary antibodies used were goat anti-mouse Alexa Fluor 488 (Invitrogen) and goat anti-rabbit Alexa Fluor 546 (Invitrogen). Embryos were mounted in Aquapolymount (Polysciences). Embryos imaged in the mid-sagittal plane were manually oriented [64 (link)], and embryos imaged in cross-section were manually sectioned [65 (link)]. Imaging was performed with a Nikon Ti-E microscope with an A1 confocal system. Images are shown in Fig 1.