In situ Hybridization and Phalloidin Staining

In situ hybridization was performed as described previously (Kraus et al., 2016 (link)) with a single change: the embryos were fixed for 1 h at room temperature in 4%PFA/PBS, washed several times in PTw (1× PBS and 0.1% Tween 20), then in 100% methanol and finally stored in 100% methanol at −20°C. Digoxigenin-labelled RNA probes were detected with anti-digoxigenin-AP Fab fragments (Roche) diluted 1:4000 in 0.5% blocking reagent (Roche) in 1× MAB. After unbound antibody was removed by a series of ten PTw washes of 10 min each, the embryos were stained with a mixture of NBT/BCIP, embedded in 86% glycerol and imaged using a Nikon 80i compound microscope equipped with the Nikon DS-Fi1 camera. For phalloidin staining, the embryos were fixed in 4%PFA/PTwTx (1× PBS, 0.1% Tween 20 and 0.2% Triton X-100) for 1 h at room temperature, washed five times with PTwTx, incubated in 100% acetone pre-cooled to −20°C for 7 min on ice and washed three more times with PTwTx. 2 µl of phalloidin-AlexaFluor488 (ThermoFisher) was added per 100 µl PTwTx, and the embryos were stained overnight at 4°C. After eight 10-min washes with PTwTx, the embryos were gradually embedded in Vectashield (Vector labs) and imaged with the Leica SP8 CLSM.