A11034

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 ].

After fixation samples were incubated in blocking/permeabilization buffer consisting of 1X TBS with 10% normal goat serum (NGS) and 0.1% Tween 20. Following blocking, samples were incubated with primary antibodies in 1x TBS with 2% NGS and 0.1% Tween 20. Primary antibodies were used as follows: monoclonal mouse anti-puromycin (clone 12D10, MilliporeSigma, catalog #MABE343, RRID:AB_2566826) at 1:500, human anti-ribosomal P (Immunovision, catalog #HPO-0100) at 1:3000, and rabbit anti-HA (Abcam, catalog #ab9110, RRID:AB_307019) at 1:500 – for 60 min at room temperature. Samples were then washed three times with 1X TBS. Next, samples were incubated with secondary antibodies in 1X TBS with 2% NGS and 0.1% Tween 20 for 30 min at room temperature. Secondary antibodies were used as follows: goat anti-mouse Alexa647 (Invitrogen, catalog #A-21236, RRID:AB_141725) at 1:1000, goat anti-rabbit A488 (Invitrogen, catalog #A-11034, RRID:AB_2576217) at 1:1000, and donkey anti-human Cy3 (Jackson ImmunoResearch, catalog #709-165-149, RRID:AB_2340535) at 1:500. Following secondary antibody incubation, cells were washed three times with 1X TBS at room temperature, then stored in DAPI Fluoromount G (Southern Biotech, catalog #0100–20) for <24 hr at 4°C prior to imaging.