Wild-type and mutant stocks were maintained on nutrient-rich Drosophila media at 25°C. Pre-pupae were selected and maintained in humidified chambers until dissection at 40 h after puparium formation (APF) (DeAngelis and Johnson, 2019 (link)). Rabbit anti-Cno (1:500) and chicken anti-GFP (1:8000, Abcam #13970) followed by Alexa-Fluor-488-conjugated secondary antibodies (Jackson ImmunoResearch #711-545-152 or #703-545-155) were used to detect Cno, CnoWT-GFP and cnoΔDIL-GFP, and retinas imaged with a Leica DM5500 B fluorescence microscope. We performed dissections in triplicate, with 5–10 pupae of each genotype dissected each time. Patterning errors were scored in retinas from one representative dissection of three carried out in triplicate, as previously described (Johnson and Cagan, 2009 (link)). Analyses spanned 9–15 eyes for each genotype, with 110 data points (ommatidia) per genotype. Significance was assessed using unpaired t-tests with Welch’s correction (two-tailed P-value). Image files were processed for publication using Adobe Photoshop.
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