Ofn25 60 objective

Dechorionated, formaldehyde-fixed, methanol-devittellinized embryos were fluorescently stained using standard methods (see Supplemental Experimental Procedures for a list of antibodies used). Embryos were mounted in 70% glycerol/PBS. Fluorescent mRNA in situ hybridization was performed as described, with digoxigenin labeled probe (Santiago et al., 2014 (link)). fra antisense probe was transcribed from linearized cDNA cloned into pBluescript. Fluorescence quantification was performed as described (Santiago et al., 2014 (link)). Images were acquired using a spinning disk confocal system (Perkin Elmer) built on a Nikon Ti-U inverted microscope using a Nikon OFN25 60× objective with a Hamamatsu C10600-10B CCD camera and Yokogawa CSU-10 scanner head with Volocity imaging software. Max projections were generated, cropped and processed using ImageJ.

Images of embryos and S2R+ cells were acquired using a spinning disk confocal system (Perkin Elmer) built on a Nikon Ti-U inverted microscope using a Nikon OFN25 60× objective with a Hamamatsu C10600-10B CCD camera and Yokogawa CSU-10 scanner head with Volocity imaging software. Images were processed using ImageJ. When scoring EW crossing, a segment was considered to have a crossing defect if one or both bundles of EW axons failed to reach the midline. When scoring ap crossing, a segment was considered to have an ectopic cross if it contained at least one continuous projection that extended across the midline and reached the lateral bundle of ap axons on the contralateral side. comm expression was scored using Volocity imaging software. Embryos expressed UAS-Tau-Myc-GFP and EW or ap neurons were identified by anti-Myc immunostaining. If the cell body of a neuron could be detected by the in situ signal, that neuron was scored as positive. Crossing and comm expression were scored in EW neurons at stage 14 and in ap neurons at stages 16-17. For all analyses, segments A1-A7 were scored. Midline crossing phenotypes and comm mRNA expression were scored blind to genotype whenever possible.