Fig. 4. Robo protein levels are reduced in lola mutant embryos. (A) Outline of the two-color ratio method for quantification of immunofluorescence. lola mutant embryos and their non-mutant siblings were collected and fixed 10-11 hours after egg-laying (AEL) and doubly stained with mouse anti-Robo and rabbit anti-HRP. Antibodies were detected by incubating with the indicated fluorescent secondary antibodies and using the confocal microscope to collect a stack of image slices across the entire CNS. Integrated fluorescence intensities for each CNS were determined using NIH Image 1.62 to perform an average (i.e. summation) projection of the image stack; manually outlining the CNS in the projected image, and integrating the fluorescence signals for both chromophores within the outline. Fluorescence intensity comparisons were made between embryos from a single embryo collection and staining, with data collected from a single slide in a single confocal session. (B,D) Wild-type embryo; (C,E) homozygous lola^ORC46 embryo. (B,C) Anti-HRP reference signal (visualized with Texas Red-conjugated secondary antibody); (D,E) Anti-Robo experimental signal (visualized with FITC secondary). For purposes of presentation, the intensities of the wild-type and mutant reference signals have been approximately matched and the cognate Robo signals adjusted accordingly. Quantification of the intensity ratios in this example show the Robo/HRP ratio to be decreased 40% in the mutant embryo when compared with the wild type.

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