Whole-mount Drosophila embryo immunostaining and fluorescent in situ hybridization (FISH) were performed as previously described (Schor et al., 2018 (link)). Fixation of overnight embryo collections was carried out in 4% formaldehyde (from a 16% formaldehyde ultra-pure methanol free stock (Polysciences #18814-10)) for 20 minutes. Immunostaining was performed with the following primary antibodies and dilutions: rat anti-Tropomyosin (1:4000) (Babraham #P6694), mouse anti-Fasciclin III (1:500) (DSHB #7G10), chicken anti-beta-Galactosidase (1:500) (Abcam #ab9361), rabbit anti-Mef2 (1:200) (Furlong lab) and rabbit anti-Biniou (1:200) (Furlong lab). Alexa Fluor conjugates (Thermo Fisher Scientific) were used as secondary antibodies (1:500). Digoxigenin-labeled RNA in situ probes for luna, Nk7.1, and CG14655 were prepared from corresponding EST clones using DIG RNA Labelling Mixture (Roche #11277073910) and the fluorescent detection of mRNA expression was performed using a Tyramide Signal Amplification (TSA) kit (Perkin Elmer #NEL753001KT). Stained embryos were mounted in ProLong Gold Antifade reagent (Thermo Fisher Scientific # P36931) and imaged with a Zeiss LSM780 confocal microscope using a Plan Apochromat 20x/0.8 objective. Images were then visualized in Fiji (Schindelin et al., 2012 (link)).
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