Computational protocol: Age-associated increase of the active zone protein Bruchpilot within the honeybee mushroom body

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Protocol publication

[…] Labeled brain slices were scanned with a confocal laser-scanning microscope (TCS SP2, Leica Microsystems, Wetzlar, Germany) equipped with an argon laser (488 nm), a helium-neon laser (633 nm) and a green helium-neon laser (543 nm). Optical sections were taken at a format of 1024 x 1024 pixels using a 10x air objective (HC PL APO, NA/0.4, Leica Microsystems, Wetzlar, Germany) for overview images and a 63x oil-immersion objective (HCX PL APO, NA/1.32, Leica Microsystems, Wetzlar, Germany) for images with higher magnification. Channels of triple-labeled preparations were merged with the use of pseudocolors using Zeiss LSM Image Browser (Version 3.2.0.115, Carl Zeiss Microscopy GmbH, Jena, Germany). As the staining patterns were similar in different preparations of the same experimental group, figures show representative examples.For analyzing anti-BRPlast200 and anti-SYNORF1 signals in the MBs, we chose one slice per bee where the central complex and the medial lobes of the MBs were present to ensure scanning of similar regions in each bee brain. Optical sections were taken in one optical plane from both medial MB calyces with the 63x objective. Microscope and scanning settings were kept identical for comparative brain slices of other bees. Digital images were further processed and quantified using ImageJ (ImageJ version 1.46m, Wayne Rasband, National Institutes of Health, USA, http://imagej.nih.gov/ij).To measure anti-BRPlast200 and anti-SYNORF1 signals, we placed two regions of interest (ROIs) in the dense collar and two ROIs in the lip region of both medial calyces in the scanned images. Each ROI covers a square area of 400 μm2 (86 x 86 pixels, pixel size 232 nm). The signal intensity of each pixel per ROI was measured separately for the anti-BRPlast200 and the anti-SYNORF1 staining, i.e. for each channel. Next, the lowest intensity value of a channel measured per ROI was subtracted from all other values in this ROI. Each of these background corrected values was normalized to the highest calculated value in its ROI to obtain relative values between zero and one. To detect those pixels indicating the presence of an antibody staining, each relative value that was higher than an antibody- and region-specific threshold was defined as signal (anti-BRPlast200-positive or anti-SYNORF1-positive pixels). This threshold was defined as the sum of a constant, antibody- or dye-specific value (0.1 for Alexa Fluor 546 Phalloidin, 0.05 for anti-SYNORF1, and 0.15 for anti-BRPlast200, constant values were set by the experimenter) and the median of all values in one ROI. In doing so, potential discrepancies in value intensities between different ROIs based on the staining method and between sample differences were taken into account. For the quantification of anti-SYNORF1- and anti-BRPlast200-positive pixels in lip and collar of 1-, 8-, 15-, 29- and 43-day-old bees, we calculated the median number of anti-BRPlast200- and anti-SYNORF1-positive pixels per ROI for collar and lip of each bee and the ratio between the two medians. [...] The values from quantitative Western blot analyses and from the quantification of antibody signal intensities were in most cases non-normally distributed. Therefore groups were tested with the Kruskal-Wallis test and the two-tailed Mann-Whitney U test for post-hoc comparisons. The alpha level was set to 0.05 and was Bonferroni-Holm corrected. All statistical analyses were performed with Statistica 12 (StatSoft, Tulsa, OK, USA). […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Apis mellifera, Drosophila melanogaster