Axio zoom v16 fluorescence stereomicroscope

Live and whole-mount hybridized embryos were photographed on agarose-coated dishes using either an epifluorescence Leica MZ16 F stereomicroscope (1X Plan Apo objective, NA0.141) (Leica, Wetzlar, Germany) equipped with digital camera or an Axio Zoom.V16 fluorescence stereomicroscope (Zeiss). Phalloidin-stained embryos were acquired on an Axiovert 200M fluorescence microscope (Zeiss) equipped with ApoTome.2 to enhance resolution. Evaluation of ISV defects was carried out on developing vessels in the region of the trunk above the prolongation of the yolk, as indicated in Figure 4A.
Confocal analysis of filopodia was performed using a LSM510 laser scanning microscope (Zeiss). To this purpose, 26–28 hpf embryos were fixed overnight with a PBS-based solution containing 1% PFA, 0.1% glutaraldehyde and 3% sucrose and mounted on glass slides with Mowiol 4.88 (Sigma). For consistency reasons and in order to minimize stage-related discrepancies, filopodia evaluation was carried out on the ISV pair in which the first vessel had already reached the roof of the trunk and the adjacent vessel was still growing up.

For each embryo collection, and after dechorionation, a representative embryo sample was collected from the total pool and fixed in a scintillation flask, using a solution containing one volume of 4% formaldehyde in PBT and four volumes of heptane, for 20 min at 100 rpm. The lower aqueous phase solution was subsequently removed, 4 mL of methanol was added and embryos were shaken vigorously for 1 min. Embryos were then collected from the bottom of the scintillation flask, washed twice with methanol, and frozen at −20°C in methanol. To rehydrate the embryos, they were washed for 5 min each, with 3:1, 1:1, and 1:3 mix solutions of methanol:PBT. Embryos were subsequently washed twice in PBT and incubated with 1:5000 Sytox green (Invitrogen), supplemented with 5 µg/mL RNase A (Sigma-Aldrich) in PBT for 15 min. After washing with PBT, embryos were mounted in fluorescence mounting medium (Dako) and examined in a Zeiss AxioZoom V16 Fluorescence Stereo Microscope for image acquisition and embryo staging. Images were processed using ImageJ software (NIH).