Computational protocol: External and circadian inputs modulate synaptic protein expression in the visual system of Drosophila melanogaster

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Protocol publication

[…] In each experiment, the images of the lamina of flies decapitated at different time points were collected at non-saturated settings, using identical image acquisition parameters. The changes in the level of fluorescence of BRP, SYN, and DLG-specific immunolabeling (brightness), corresponding to the changes in the amount of the given protein (BRP, SYN, or DLG), were quantified using ImageJ software (NIH). In each experiment the intensity of fluorescence was measured in the cartridges of the distal part of the cross-sectioned lamina (the second and the third row of cartridges from the lamina cortex) (Figure ), as represented by the Mean Gray Value, defined by the sum of the gray values of all pixels in the selected area, divided by the number of pixels within the selection. In the ImageJ software, the range of gray values in 8-bit images is divided into 256 bins. For each time point (ZTs or CTs) we collected data from 7 to 12 individuals. The background signal was subtracted. [...] All statistical tests were performed using the Statistica v.10 (StatSoft, Inc.). The significance of differences observed between two independent groups (e.g., day and night) were statistically analyzed using the T-test or Mann–Whitney Test, depending on the result of the Shapiro–Wilk W-Test. Statistical significance across groups, ZT or CT time points, was analyzed using One-Way ANOVA followed by a Tukey post-hoc Test or the nonparametric counterpart of ANOVA—Kruskal–Wallis Test (one-way test), followed by the Multiple Comparison Test. To assess differences between LD 12:12 and DD we performed the Two-Way ANOVA regarding the light conditions and the time points (ZTs and CTs) as two key factors.Immunolabeling quantification data are shown as a percentage of the highest value (100%) that was obtained in a given experiment (means ± SD).For visualization of qPCR data, gene expression was normalized on an arbitrary scale, where the ZT1 time point was set to 1.00 (means ± SE). […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Drosophila melanogaster
Diseases Intervertebral Disc Degeneration