Mosaic tissues were obtained using the FLP/FRT system with hsFlp. Flies were heat-shocked for 60 min at 48 h and 72 h after egg deposition. Pupae were staged by collecting white prepupae 3 days after the first heat-shock and incubating at 25°C for the indicated times. Pupal wings and retinas were fixed in 8% and 4% paraformaldehyde in PBS, respectively, for 30 min (larval wing discs in 4% paraformaldehyde in PBS for 30 min), washed three times with PBS, permeabilised with PBT (PBS+0.3% Triton x100), blocked with PBT+1% BSA, and immunostained using the indicated primary antibodies in PBT+1% BSA at 4°C overnight and secondary antibodies for 2 h at room temperature.

Primary antibodies used were rabbit anti-Rab5, anti-Rab7, anti-Rab11 (1:2000, gifts from Akira Nakamura, Kumamoto University, Japan; Tanaka and Nakamura, 2008), guinea pig anti-Sec15 and anti-Hrs (1:1000, a gift from Hugo Bellen; Lloyd et al., 2002; Mehta et al., 2005), rat anti-Ed (1:1000, a gift from Jui-Chou Hsu, National Chiao Tung University, Taiwan; Wei et al., 2005), mouse anti-Sec5 (22A2) antibody (1:200, a gift from Thomas Schwartz, Harvard Medical School, Boston, MA, USA; Murthy et al., 2003). The rat anti DE-cadherin (1:100, developed by T. Uemura, Kyoto University, Japan), mouse anti-Arm (1:100, developed by E. Wieschaus, Princeton University, NJ, USA) and anti-Fmi #74 (1:20, developed by T. Uemura) were obtained from the Developmental Studies Hybridoma Bank. Secondary antibodies used were rhodamine red-X donkey anti-rabbit, anti-rat and anti-mouse, fluorescein isothiocyanate (FITC) donkey anti-rabbit, anti-rat and anti-mouse (Jackson ImmunoResearch), goat anti-rat Alexa 647, and goat anti-rabbit Alexa 633, all at 1:500. Fluorescence images were acquired with a Zeiss LSM780 or a Zeiss LSM880 confocal.

Antibodies against transmembrane proteins tested for accumulation in RASSF8 mutant clones were: mouse anti-Fmi (1:20, DSHB Flamingo #74), rabbit anti-Ds (1:1000, from David Strutt, University of Sheffield, UK; Strutt and Strutt, 2002), mouse anti-Roughest/IrreC (1:10, from Karl-Friedrich Fischbach, Albert-Ludwigs-University Freiburg, Germany; Schneider et al., 1995), mouse anti-Notch-ICD (1:100, DSHB C17.9C6; deposited by S. Artavanis-Tsakonas, Harvard Medical School, Boston, MA, USA), rat anti-Crb (1:1000, from Franck Pichaud, University College London, UK; Walther et al., 2016), rat anti-Fat (1:1000, from Helen McNeill, Washington University School of Medicine, St Louis, MO, USA), rabbit anti-EGFR (1:1000, from Erika Bach, New York University Grossman School of Medicine, USA), rat anti-SNS (1:1000, from Susan Abmayr, Stowers Institute for Medical Research, Kansas City, MO, USA; Bour et al., 2000), rat anti-Hibris (1:1000, from Tetsuya Tabata, The University of Tokyo Bunkyo-ku, Japan; Sugie et al., 2010) and rat anti-Kirre (1:1000, from Susan Abmayr; Galletta et al., 2004).