Spe live confocal microscope

Fluorescent tracing of VDA-derived HSPCs colonizing the CHT was done using the tg(kdrl:Dendra2) line as described before [32 (link), 33 (link)] with a Leica SP5 confocal microscope with a ×20 dry objective. At 28 hpf an area of ~40 × 750 nm around the VDA, parallel to the yolk sac extension was photoconverted. The 405 nm UV laser intensity and exposure time were optimized for strong Dendra2-conversion without cell damage. After photoconversion embryos were transferred to E3 medium and at 50–60 hpf their CHT areas were imaged on a Leica SPE Live confocal microscope using a ×20 dry objective. To exclude bleed-through of Dendra-green, red channel detection was set stringently (630–680 nm).

To photograph somite boundaries, 18–19.5 hpf embryos were dechorionated and laterally aligned in conical depressions that fit the yolk, in an agarose pad (Sigma, 2% in E3) cast in a petri dish (Falcon, 50 mm x 9 mm) and topped up with E3. Photomicrographs were taken on an Olympus MVX10 microscope equipped with an Olympus DP22 camera.
For imaging the notochord, embryos were mounted in 0.5% low melting point agarose in a culture dish with a cover slip replacing the bottom. Fluorescent imaging was performed with a Leica SPE 'live' Confocal Microscope, Leica SP8 confocal microscope and a PerkinElmer Ultraview VoX spinning disk microscope using a 10x or 20x objective with digital zoom. Usually, z-stacks with intervals of approximately 2 µm were captured and were then flattened by maximum projection in ImageJ. For photoconversion of whole entpd5:kaede embryos, the green Kaede fluorophore was photoconverted using a Leica fluorescence microscope by 15–30 min exposure through a UV light bandpass filter (360/40 nm, 100 W mercury lamp). For bone stains an Olympus S2 × 16 microscope coupled to a Leica DFC420c camera was used. Photomicrographs were stitched with the pairwise stitching plugin in Fiji (RRID:SCR_002285) (Preibisch et al., 2009 (link)).