Goat anti gfp fitc

The following antibodies were used: Rabbit Phospho-histone H3 (Cell Signalling #9701, 1:200), rabbit anti-Engrailed (Santa Cruz Biotechnology d-300; 1:200), goat anti-GFP-FITC (Abcam ab6556, 1:500), guinea pig anti-Sqh-1P (1:100, (Zhang and Ward, 2011 (link))) (called MRLC-1P in this paper), mouse anti-phospho-Tyrosine (Cell signaling #9411; 1:1000), mouse anti-Wingless (DSHB 4D4; 1:50); mouse anti-Dlg (DSHB 4F3; 1:500) Rabbit anti-Pins (Izumi et al., 2006 (link)) (1:1000, a gift from F. Matsuzaki), rabbit anti-Mud(Izumi et al., 2006 (link)) (1:200, a gift from F. Matsuzaki). Secondary antibodies conjugated to fluorescent dyes were obtained from Jackson ImmunoResearch Laboratories, Invitrogen and Life Technologies. Cell nuclei were stained using DAPI (Sigma-Aldrich).

Neuronal cultures were fixed with 4% PFA. Coverslips were then rinsed × 3 in PBS and incubated at room temperature for 1 h in PBS, 0.2% Triton X-100, 4% donkey serum (Sigma-Aldrich), followed by primary antibody (1 h, PBS, 0.2% Triton X-100) and then secondary antibody before mounting in ProlongGold Antifade Reagent (Molecular Probes). Primary antibodies: mouse anti-Myc (1:500; Sigma-Aldrich, #M4439, clone number 9E10), rabbit anti-Myc (1:500; Millipore, #06–549), rat anti-Ctip2 (1:500; Abcam, #ab18465), rabbit anti-MAP2 (1:500; Millipore, #AB5622), goat anti-GFP-FITC (1:1000; Abcam, #ab6662). Secondary antibodies were conjugated to Alexa Fluor 488, 546, 568, or 647 (1:1000; Molecular Probes). Images were acquired on a Diskovery spinning disk microscope with a 60X objective, numerical aperture: 1.4 (Nikon), with a z-step of 130 nm (xy pixel size: 93 nm) and deconvolved using Huygens Professional v16.10 software (Scientific Volume Imaging).