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. 2017 Oct 6;6:e26519. doi: 10.7554/eLife.26519

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© 2017, Qian et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 4. — (A) Schematic illustration of different brain regions regulating sleep (above). dFB, dorsal fan-shaped body; DN1, dorsal neurons 1; lLNv, large ventral lateral clock neurons; MB, mushroom body; PI, pars intercerebralis. Sleep in flies expressing different Gal4-driven 5HT2b RNAi lines (below). (B) Brain of UAS-mCD8GFP; 23E10-Gal4 flies, immunostained with the anti-GFP antibody (green) and the nc82 antibody (red). (C) Brain of UAS-DenMark,UAS-sytGFP; 23E10-Gal4 fly, immunostained with the anti-GFP antibody (green), the anti-RFP antibody (red) and the neuropil marker nc82 antibody (blue). (D and E) Intersectional neurons of 23E10 and 5HT2b. Brain of 23E10-LexA/UAS-FRT-mCD8GFP-FRT; LexAop-Flp/Trh::Gal4 fly, immunostained with the anti-GFP antibody (green) and the neuropil marker nc82 antibody (red). Two intersectional neurons were labeled (E). (F–H) One pair of 5HT2b and 23E10 co-stained neurons. 5HT2b-LexA driven LexAop-tdTomato labelled 5HT2b neurons after anti-RFP immunostaining, and 23E10-Gal4 driven UAS-stingerGFP labelled 23E10 neurons after anti-GFP immunostaining. (F1, G1, and H1) Whole-mount staining. (F2, G2, and H2) Cell bodies of two co-stained neurons. NC82 fluorescences blue. Arrows indicate the cell bodies of co-stained neurons. (I–K) Immunostainings of 5HT2b^sfGFP and serotonin. 5HT2b^sfGFP labelled green, 5-HT labelled red and NC82 labelled blue as background. (I1, J1, and K1) Whole-mount staining. (I2, J2, and K2) Slice of FB region staining. (L–N) Immunostaining of 5HT2b^sfGFP at dFB and serotonin. UAS-5HT2b^sfGFP driven by 23E10 labelled green, 5HT labelled red and NC82 labelled blue as background. (L1, M1, and N1) Whole-mount staining. (L2, M2, and N2) Slice of FB region.

Figure 4—figure supplement 1. — (A) Schematic illustration of different brain regions regulating sleep (above). dFB, dorsal fan-shaped body; DN1, dorsal neurons 1; lLNv, large ventral lateral clock neurons; MB, mushroom body; PI, pars intercerebralis. Sleep in flies expressing different Gal4-driven 5HT2b RNAi lines (below). (B) Brain of UAS-mCD8GFP; 23E10-Gal4 flies, immunostained with the anti-GFP antibody (green) and the nc82 antibody (red). (C) Brain of UAS-DenMark,UAS-sytGFP; 23E10-Gal4 fly, immunostained with the anti-GFP antibody (green), the anti-RFP antibody (red) and the neuropil marker nc82 antibody (blue). (D and E) Intersectional neurons of 23E10 and 5HT2b. Brain of 23E10-LexA/UAS-FRT-mCD8GFP-FRT; LexAop-Flp/Trh::Gal4 fly, immunostained with the anti-GFP antibody (green) and the neuropil marker nc82 antibody (red). Two intersectional neurons were labeled (E). (F–H) One pair of 5HT2b and 23E10 co-stained neurons. 5HT2b-LexA driven LexAop-tdTomato labelled 5HT2b neurons after anti-RFP immunostaining, and 23E10-Gal4 driven UAS-stingerGFP labelled 23E10 neurons after anti-GFP immunostaining. (F1, G1, and H1) Whole-mount staining. (F2, G2, and H2) Cell bodies of two co-stained neurons. NC82 fluorescences blue. Arrows indicate the cell bodies of co-stained neurons. (I–K) Immunostainings of 5HT2b^sfGFP and serotonin. 5HT2b^sfGFP labelled green, 5-HT labelled red and NC82 labelled blue as background. (I1, J1, and K1) Whole-mount staining. (I2, J2, and K2) Slice of FB region staining. (L–N) Immunostaining of 5HT2b^sfGFP at dFB and serotonin. UAS-5HT2b^sfGFP driven by 23E10 labelled green, 5HT labelled red and NC82 labelled blue as background. (L1, M1, and N1) Whole-mount staining. (L2, M2, and N2) Slice of FB region.