4.5.2 Confocal microangiography

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4.5.2 Confocal microangiography

Misato Fujita
Tokyo Institute of Technology

4.5.2.1 General information

Confocal microangiography method was developed and explained previously (Weinstein et al., 1995; Isogai et al., 2001). Since medaka embryo has large yolk ball, embedding method for injection is changed (Fujita et al., in preparation).

4.5.2.2 Preparation

1. Dilute Fluoresceinated carboxylated latex beads (0.02 to 0.04mm) (F8787, F8794, F8783; Molecular Probes) by 1:1 with 2% BSA (Sigma) in deionized distilled water and sonicate by a homogenizer.
2. Cetrifuge the beads solution for 2 min at top speed in an Eppendorf microcentrifuge (beads suspension is of usage).
3. Prepare the grass microinjection needles by grass capillaries using a vertical pipette puller.
4. Dechorionate embryos.

4.5.2.3 Microangiography

1. Anesthetize required staged embryo with 0.84% tricaine (Sigma).
2. Embed an embryo in 1% low melting temperature agarose for injection.
3. Insert needles obliquely into the sinus venosus and inject beads.
4. Reorient an injected embryo in 1% low melting temperature agarose for imaging using confocal microscope.
5. Collect vascular wiring images on the confocal microscope using the proper laser line.

4.5.2.4 Reference

Weinstein BM, Stemple DL, Driever W, Fishman MC (1995). Gridlock, a localized heritable vascular patterning defect in the zebrafish. Nat Med 1:1143-1147.

Isogai S, Horiguchi M, Weinstein BM (2001). The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Dev Biol 230:278-301.