Adult brain imaging of Drosophila melanogaster T2A-GAL4 (TG4) lines driving UAS-nls.mcherry co-stained with either neuronal (elav) or glial (repo) nuclear markers. Slices are combined to visualize a coronal sections throughout the brain and gene-reporting of indicated fly genes.
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All TG4 lines are crossed with UAS-nls.mCherry (3rd chromosome) or UAS-nls.GFP (3rd chromosome) at room temperature. Note that the nls.GFP that is being used here is prone to leak outside the nucleus while nls.mCherry is retained in the nucleus. The brains of mCherry/GFP positive third instar larvae and 3-5 days old adult flies were dissected in 1X phosphate-buffered saline (PBS). Adult brains were fixed immediately in 4% paraformaldehyde (PFA) and incubated at 4°C overnight (o/n) on a shaker. Next day these brains were post-fixed with 4% PFA with 2% Triton-X in PBS (PBST), kept in a vacuum container for an hour to get rid of the air from the tracheal tissue also make the tissue more permissive. Fixative was replaced every 10 minutes during this post-fixation step. Larval brains were fixed for 50 minutes on a rotator at room temperature. After thorough washing with PBS with 0.2% Triton (PBTX) both adult and larval brains were incubated with primary antibodies overnight (o/n) at 4°C on a shaker. The sample were extensively washed with 0.2% PBTX before secondary antibodies were applied at room temperature for 2 hours. Samples were thoroughly washed with PBST and mounted on a glass slide using Vectasheild (Vector Labs, H-1000-10). Primary antibodies used: Mouse anti-repo (DSHB: 8D12) 1:50, Rat anti-elav (DSHB: 7E8A10) 1:100, Goat anti-GFP (abcam: ab6662) 1:500. Secondary antibodies used: Anti-mouse-647 (Jackson ImmunoResearch: 715-605-151) 1:250, Anti-rat-Cy3 (Jackson ImmunoResearch: 712-165-153) 1:500. The samples were scanned using a laser confocal microscope (Zeiss LSM 880) with a 20X objective, and images were processed using ZEN (Zeiss) and Imaris (Oxford Instruments) software. Co-localization between mCherry and Elav or mCherry and Repo was performed with default thresholds in Imaris.