Beetles were reared according to The Beetle Book (http://wwwuser.gwdg.de/~gbucher1/tribolium-castaneum-beetle-book1.pdf) at 32°C. Eggs were dechorionated in 0.5% bleach and lined up on a microscope slide for upright microscopy or on a glass-bottomed Petri dish (MatTek) that had a window cut in the side of the dish to allow injection for inverted microscopy. Gaps were left between neighboring eggs for efficient gas exchange. Eggs were covered with Voltalef 10S Halocarbon oil. The needle was inserted into the egg at the anterior pole and the tip of the needle was moved into the center of the egg for injection. Capped mRNAs were injected at 0.5-3 μg/μl in injection buffer (5 mM KCl, 10 mM NaH2PO4). To monitor the kinetics of fluorescence labeling, we injected H2B-RFP and/or GAP43-YFP mRNAs into eggs 4-6 hours after egg lay (AEL) at 32°C, when nuclei migrate towards the egg surface and the cellularized blastoderm begins to form. In all other applications, injections were performed in eggs 2-3 hours AEL at 32°C to achieve maximum diffusion and homogeneous labeling. Following injection at room temperature, eggs were transferred back to 32°C and incubated in a dark and humid environment. Under these conditions, Tribolium embryos developed normally, but hatching rates were decreased, probably due to egg immersion in halocarbon oil. Microinjections caused a developmental delay of 30-60 minutes, as judged by the time of initiation of the posterior amniotic fold between injected and non-injected embryos. Live imaging was carried out on an inverted Leica SP5 confocal at 32°C. Image stacks of about 40 focal planes were taken with a 20×/0.7NA multi-immersion objective or a 40×/1.3NA oil-immersion objective at 2.5- or 5-minute intervals. Processing of confocal stacks was carried out with Fiji (Schindelin et al., 2012 (link)) and cell tracking with MTrackJ (Meijering et al., 2012 (link)).