Fruitflies help researchers decode genetic link to Alzheimer'sdisease WEHI_research SciReports
Strains were reared on molasses-based Drosophila medium. Briefly, 1.4 kg of molasses is mixed in 3 l of water and 900 g of yeast mixed with 3 l of water before pouring into a 30 l metal pot and placing on a gas burner. The mixture was brought to 100 °C with occasional stirring. 630 g of semolina was mixed with 3 l of water and added. Upon the mixture obtaining a homogeneous consistency, it was cooled to 80 °C prior to the addition of 138 ml Acid Mix and 262 ml Tegosept .
A Zeiss LSM 800 running Zen Blue software was used to visualize and analyse the eye imaginal discs. Serial Z-series were captured in 1 μm sections at 40 × magnification. Fluorophores were imaged using band-pass filters to remove cross-detection between channels and pseudo-coloured for image preparation. Confocal images in generic format were processed using Image J 1.48v, and Adobe Photoshop CS5 Version 12.0.4.I.
Tom40-HA levels were quantified using FIJI . Data points represent different eye discs with the relevant genotype. For each image, 3 μm sections were merged with maximum projection, 5 ommatidia per image were selected based on DAPI staining to identify nuclei, and the average raw Tom40-HA intensity was measured within the selection. For quantification of Mito-YFP area proportion, 5 μm sections were merged and Auto Local Threshold applied to calculate the proportion of total area.
BN-PAGE analysis of solubilised cell membranes from adult fly heads was performed at 4 °C using the Native PAGE Bis–Tris Gel System on 4–16% Bis–Tris gels using NativeMark unstained protein standards. Tagged Tom40 was detected by western blot against the HA peptide.
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