Computational protocol: Olfactory subsystems in the honeybee: sensory supply and sex specificity

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Protocol publication

[…] Brains were placed in fixative solution (4 % formaldehyde) overnight, following the staining procedure and then rinsed five times for 10 min in PBS (phosphate-buffered saline, pH 7.2). Biotin-Dextran-injected brains were stained with Alexa-488-conjugated Streptavidin (S-11226, Molecular Probes, Eugene, Ore., USA) in PBS with 0.2% Triton X (1:125) for 48 h. Subsequently, brains were again rinsed five times for 10 min in PBS. Microruby- and Biotin-Dextran-labeled brains were dehydrated in an ascending ethanol series (50, 70, 90, 95, 2× 100 %, each 10 min) and cleared and mounted in methyl salicylate (M2047, Sigma-Aldrich Chemie) for confocal microscopy. Afterwards, brains were scanned with a confocal laser-scanning microscope (Leica TCS SP2; Leica Microsystems, Wetzlar, Germany). Images were processed with AMIRA 3.1.1 and 5.4 software (Mercury Computer Systems, Berlin, Germany) and AL glomeruli in one preparation were reconstructed by using the AMIRA wrapping tool. Image stacks were further processed with ImageJ 1.46j (Wayne Rasband, National Institutes of Health, Befesta, Md., USA). To count the glomeruli in all other successful mALT stainings (that were not three-dimensionally reconstructed in detail), we marked glomeruli in image stacks by using the segmentation editor implemented in ImageJ. Contrast and brightness were adjusted with GIMP 2.8.2 (GNU Image Manipulation Program, http://www.gimp.org) and images in figures were arranged with CorelDrawX6 (Corel, Ottawa, ON, Canada). […]

Pipeline specifications

Software tools ImageJ, GIMP
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Apis mellifera