Immunolabeling Analysis of GAL4 Lines

Immunolabelling for the analysis of GAL4 lines was performed as previously described [5 (link)–7 (link),12 ]. Either 97% TDE [53 (link)] or VECTASHIELD (VECTASHIELD®, Vector) was used as mounding medium. The employed primary antibodies were the rabbit anti-GFP (1:1000; Invitrogen; A11122) or rat anti-N-cadherin (DN-EX #8; 1:100; Developmental Studies Hybridoma Bank). The employed secondary antibodies were the cross-adsorbed secondary antibodies to IgG (H+L): AlexaFluor-488 goat anti-rabbit (1:1000; Invitrogen; A11034) or Cy3 goat anti-rat (1:200; Jackson Labs). Optical sections of whole-mount brains were sampled with a confocal microscope (Olympus FV1200). Confocal stacks were analyzed with the open-source software Image-J (National Institute of Health) and Fiji [54 ].