Microangiography of zebrafish embryos and larvae was performed essentially as described previously (Weinstein et al., 1995), with modifications as noted below.

• FISH: Zebrafish husbandry, embryo collection, proteinase dechorionation, and embryo and larva maintenance were performed as described in The Zebrafish Book (Westerfield, M. 1995, University of Oregon Press, Eugene, OR- available online here). The use of albino mutant lines improved visualization of many vascular beds. PTU treatment was also used to inhibit pigment formation in wild-type animals (see The Zebrafish Book for PTU treatment protocol).
• MATERIALS: Fluoresceinated carboxylated latex beads were obtained from Molecular Probes. 0.02-0.04 micron sized beads are used, either yellow-green (cat # F8787), red-orange (cat # F8794), or dark red (cat # F8783). These were detected by the 488, 568, and 647 nm laser lines from the Krypton-Argon laser on our BioRad MRC-1024 (the same laser used on many conventional confocal microscopes). 1 mm OD glass capillaries were obtained from World Precision Instruments (cat # TW100-4 or TW100F-4 for glass without or with an internal filament). Glass microneedles were prepared from 1 mm capillaries using a Kopf vertical pipette puller (approximate settings: heat=12, solenoid=4.5). Needles were broken open with a razor blade just behind their tip to give an opening of approximately 5-10 microns in width. Holding pipettes were prepared from 1 mm capillaries by carefully partially melting one end of the capillary with a bunsen burner, such that the opening was narrowed to approximately 0.2 mm (slightly smaller for younger embryos, slightly larger for older larvae). Photographic images of the end of a microneedle and the tip of a holding pipette are shown for comparison. Microneedles and holding pipettes were attached to pipette holders (holder, WPI cat # MPH6912; adapter for holder and tubing to attach to picopump, WPI cat # 5430). Microinjection was performed using air pressure provided by a World Pro ecision Instruments Pneumatic Picopump (catalog # PV820). The holding pipettes and their holders were attached via mineral-oil filled tubing (Stoelting Instruments, Clay-Adams cat # 427415) to a manual microsyringe pump (Stoelting Instruments cat # 51222, with 25 microliter syringe). Holding pipettes and microneedles and their associated holders and other equipment were arranged on either side of a Leica MZ12 dissecting microscope as diagrammed here. Photographic images of a typical arrangement are shown here (wide view) and here (closer view) A fluorescence attachment on a separate MZ12 was used to monitor the success of the microinjection procedure. Images were collected on a BioRad MRC 1024 confocal microscope attached to a Zeiss Axioplan-2.
• INJECTION PROTOCOL: Embryos were collected, dechorionated using proteinase, and incubated in embryo medium to the desired developmental stage. Fluoresceinated carboxylated latex beads were prepared for injection as follows: bead suspension as supplied was diluted 1:1 with 2 % BSA (Sigma) in deionized distilled water, sonicated approximately 25 cycles of 1" each at maximum power on a Branson sonifier equipped with a microprobe, and subjected to centrifugation for 2 minutes at top speed in an Eppendorf microcentrifuge. Suspension was used to backfill glass microneedles for injection. Dechorionated embryos in embryo medium were anesthetized with tricaine (Sigma) as per The Zebrafish Book. 1-3 day old embryos were held ventral side up for injection using a holding pipette applied to the side of the yolk ball, with suction applied via a microsyringe driver. Care was taken not to allow the holding pipette to rupture the yolk ball. 4-7 day old larvae were held ventral side up for injection by embedding in 0.5% low melting temperature agarose. For 1-3 day old embryos a broken glass microneedle was inserted obliquely into the sinus venosus (as diagrammed here). For 4-7 day old larvae a broken glass microneedle was inserted through the pericardium directly into the ventricle. After microneedle insertion, many (20+) small boluses of bead suspension were delivered over the course of up to a minute. Smaller numbers of overly large boluses frequently resulted in temporary or permanent cardiac arrest.
• IMAGE COLLECTION: Embryos were allowed to recover from injection briefly (1 minute) in tricaine-free embryo media, after which the embryos were rapidly mounted in 5% methyl cellulose (Sigma) or low-melt agarose (both in embryo media with tricaine) and imaged on the confocal microscope using the appropriate laser line. Although the fluorescent beads distributed uniformly throughout the vasculature of the embryo within minutes, they began to be phagocytosed by and concentrate in selected cells lining the vessels shortly thereafter (cf "tail reticular cells" in The Zebrafish Book). Because of this, specimens were scanned as rapidly as possible, generally within 15 minutes after injection. Between 20 and 40 frame-averaged (6 frames) optical sections were collected with a spacing of 2-5 µm between sections, depending on the magnification (smaller spacing at higher magnifications). Three-dimensional reconstructions of saved image stacks were generated using either the BioRad confocal software or the Metamorph software package (Universal Imaging Inc.).