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4 protocols using dylight 405 donkey anti mouse

1

Multicolor Immunofluorescence Labeling

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Primary antisera used were rabbit anti-Vasa (R. Lehmann laboratory), mouse monoclonal anti-Hts (1B1, DSHB)39 , mouse monoclonal anti-Orb (4H8, DSHB)40 , mouse monoclonal anti-HA (abcam ab130275), chicken anti-GFP (Aves Labs, GFP-1020), mouse monoclonal anti-ATP5A [15H4C4] (abcam ab14748), mouse anti-PDH E1 alpha (Abcam, ab110334) and rabbit anti-phoshpo-PDH E1alpha (S293) (Millipore, AP1062). Secondary antibodies were DyLight™ 405 donkey anti-rabbit, DyLight™ 405 donkey anti-mouse, Cy3 donkey anti-mouse, all from Jackson ImmunoResearch, and Alexa fluor 488 goat anti-chicken from ThermoFisher Scientific.
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2

Immunofluorescence of Hippocampal Neurons

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Hippocampal slices were fixed in 4% paraformaldehyde for 1 hr at room temperature. The slices were blocked with 5% normal donkey serum (DNS; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and 0.01%Triton X-100 in PBS for 2 hr. The primary antibodies were diluted into a 10% DNS/0.005%Triton X-100 solution and applied to slices at 4°C overnight. They were rabbit anti-TREK-1 (1:1000, Alomone Labs, Jerusalem, Israel) and mouse anti-NeuN (1:1000; Abcam, Cambridge, MA, USA). The secondary antibodies were Cy3 donkey anti-rabbit (1:1000) and DyLight 405 donkey anti-mouse (1:1000) (Jackson ImmunoResearch Laboratories). Immunofluorescence images were obtained from a confocal microscope (TCS-SL, Leica Microsystems, Buffalo Grove, IL, USA).
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3

Multicolor Immunofluorescence Labeling

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Primary antisera used were rabbit anti-Vasa (R. Lehmann laboratory), mouse monoclonal anti-Hts (1B1, DSHB)39 , mouse monoclonal anti-Orb (4H8, DSHB)40 , mouse monoclonal anti-HA (abcam ab130275), chicken anti-GFP (Aves Labs, GFP-1020), mouse monoclonal anti-ATP5A [15H4C4] (abcam ab14748), mouse anti-PDH E1 alpha (Abcam, ab110334) and rabbit anti-phoshpo-PDH E1alpha (S293) (Millipore, AP1062). Secondary antibodies were DyLight™ 405 donkey anti-rabbit, DyLight™ 405 donkey anti-mouse, Cy3 donkey anti-mouse, all from Jackson ImmunoResearch, and Alexa fluor 488 goat anti-chicken from ThermoFisher Scientific.
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4

Immunostaining of Drosophila Larval and Adult Tissues

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Wandering third instar larvae were dissected and pinned open as filets in cold PBS and fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. Larvae were then permeabilized in 0.3% PBST (PBS + Triton X-100) for 15 min at room temperature with agitation, and remaining wash and antibody solutions were made in 0.3% PBST with 1% BSA. Samples were incubated in primary antibody solution overnight at 4°C followed by one quick wash then 5 × 15 min washes using 0.3% PBST before incubating in secondary antibody solution overnight at 4°C. Samples were then washed again as before, then incubated in Vectashield (#H-1000; Vector Labs) for 1 h at room temperature before mounting. Antibodies used were: (1°) anti-Repo (Mouse anti-Repo, #8D12; DSHB), Alexa Fluor 647 anti-HRP (Goat anti-HRP, #123-605-021; Jackson Labs), anti-oaz (Rabbit anti-oaz, (Corty et al., 2022 (link)), anti-GFP (Chicken anti-GFP #ab13970; Abcam); (2°) DyLight 405 Donkey anti-Mouse (#715-475-150; Jackson Labs), Alexa Fluor 488 Donkey anti-Chicken (#703-545-155; Jackson Labs), and Rhodamine Red-X Donkey anti-Rabbit (#711-295-152; Jackson Labs). After staining, larva filets were mounted in Vectashield and covered with #1.5 cover glass (#1404-15; Globe scientific) and stored at 4°C. The same protocol was used for adult wings; however, both primary and secondary incubation steps were done for 5 d at 4°C.
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