Journal: Cell Reports Methods
Article Title: A conditional strategy for cell-type-specific labeling of endogenous excitatory synapses in Drosophila
doi: 10.1016/j.crmeth.2023.100477
Figure Lengend Snippet: A quantitative analysis of postsynaptic DLG1 puncta for PNs and LNs in the DA1 glomerulus using dlg1[4K] (A) Schematic of the Drosophila antennal lobes showing presynaptic ORNs (orange) and postsynaptic PNs (magenta) in the DA1 glomerulus. (B) Representative confocal maximum projections of DA1 ORNs expressing Brp-Short-mStraw and DA1 PNs expressing DLG1-V5 and stained with antibodies against mStraw (red), V5 (green), and N-Cadherin (blue). (C) Single, high-magnification optical sections of the DA1 ORNs and PNs from (B). (D) Quantification of Brp-Short-mStraw puncta for ORNs and DLG1-V5 puncta for PNs. (E) Cumulative frequency histogram of the nearest-neighbor distance between DLG1-V5 puncta in DA1 PNs. The average NND (μ) and Cluster % of puncta are indicated. Gray traces represent individual glomeruli, and the green trace represents the aggregate average. (F) Schematic of the Drosophila antennal lobes showing presynaptic ORNs (orange) and postsynaptic LNs (blue) in the DA1 glomerulus. (G and H) Representative confocal image stacks and corresponding single, high-magnification sections of DA1 ORNs expressing Brp-Short-mStraw and multiglomerular LNs in the DA1 glomerulus expressing DLG1-V5 and stained with antibodies as in (B) and (C). (I) Quantification of Brp-Short-mStraw puncta for ORNs and DLG1-V5 puncta for LNs. (J) Cumulative frequency histogram of the nearest-neighbor distance between DLG1-V5 puncta in multiglomerular LNs in DA1, including the average (μ) and the cluster %. Traces represent individual glomeruli (gray) or the aggregate average (green). (K) Schematic of the Drosophila antennal lobes showing presynaptic PNs (magenta) and postsynaptic LNs (blue) in the DA1 glomerulus. (L and M) Representative confocal image stacks and corresponding single optical sections of DA1 PNs expressing Brp-Short-mStraw and multiglomerular LNs in the DA1 glomerulus expressing DLG1-V5 and stained with antibodies as in (B) and (C). (N) Quantification of Brp-Short-mStraw puncta for PNs and DLG1-V5 puncta for LNs. (O) Cumulative frequency histogram of the nearest-neighbor distance between DLG1-V5 puncta in the multiglomerular LNs from (L) and (M), including the average (μ) and the cluster %. Traces represent individual glomeruli (gray) or the aggregate average (green), as previously described. For all conditions, n ≥ 6 glomeruli from three brains, and 900 (E), 675 (J), or 450 (O) individual puncta. Scale bars, 5 μm.
Article Snippet: The individual values for “DistMin” and cumulative frequency histograms were obtained from Imaris and compiled in Prism 8 (GraphPad Software, Inc., La Jolla, CA).
Techniques: Expressing, Staining
GraphPad Software Inc is a verified supplier 
![A quantitative analysis of postsynaptic DLG1 puncta for PNs and LNs in the DA1 glomerulus using dlg1[4K] (A) Schematic of the Drosophila antennal lobes showing presynaptic ORNs (orange) and postsynaptic PNs (magenta) in the DA1 glomerulus. (B) Representative confocal maximum projections of DA1 ORNs expressing Brp-Short-mStraw and DA1 PNs expressing DLG1-V5 and stained with antibodies against mStraw (red), V5 (green), and N-Cadherin (blue). (C) Single, high-magnification optical sections of the DA1 ORNs and PNs from (B). (D) Quantification of Brp-Short-mStraw puncta for ORNs and DLG1-V5 puncta for PNs. (E) <t>Cumulative</t> frequency histogram of the nearest-neighbor distance between DLG1-V5 puncta in DA1 PNs. The average NND (μ) and Cluster % of puncta are indicated. Gray traces represent individual glomeruli, and the green trace represents the aggregate average. (F) Schematic of the Drosophila antennal lobes showing presynaptic ORNs (orange) and postsynaptic LNs (blue) in the DA1 glomerulus. (G and H) Representative confocal image stacks and corresponding single, high-magnification sections of DA1 ORNs expressing Brp-Short-mStraw and multiglomerular LNs in the DA1 glomerulus expressing DLG1-V5 and stained with antibodies as in (B) and (C). (I) Quantification of Brp-Short-mStraw puncta for ORNs and DLG1-V5 puncta for LNs. (J) Cumulative frequency histogram of the nearest-neighbor distance between DLG1-V5 puncta in multiglomerular LNs in DA1, including the average (μ) and the cluster %. Traces represent individual glomeruli (gray) or the aggregate average (green). (K) Schematic of the Drosophila antennal lobes showing presynaptic PNs (magenta) and postsynaptic LNs (blue) in the DA1 glomerulus. (L and M) Representative confocal image stacks and corresponding single optical sections of DA1 PNs expressing Brp-Short-mStraw and multiglomerular LNs in the DA1 glomerulus expressing DLG1-V5 and stained with antibodies as in (B) and (C). (N) Quantification of Brp-Short-mStraw puncta for PNs and DLG1-V5 puncta for LNs. (O) Cumulative frequency histogram of the nearest-neighbor distance between DLG1-V5 puncta in the multiglomerular LNs from (L) and (M), including the average (μ) and the cluster %. Traces represent individual glomeruli (gray) or the aggregate average (green), as previously described. For all conditions, n ≥ 6 glomeruli from three brains, and 900 (E), 675 (J), or 450 (O) individual puncta. Scale bars, 5 μm.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1928/pmc10261928/pmc10261928__gr5.jpg)