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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

+ 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. 
+ Cetrifuge the beads solution for 2 min at top speed in an Eppendorf microcentrifuge (beads suspension is of usage).
+ Prepare the grass microinjection needles by grass capillaries using a vertical pipette puller.
+ Dechorionate embryos.

**4.5.2.3 Microangiography

+ Anesthetize required staged embryo with 0.84% tricaine (Sigma).
+ Embed an embryo in 1% low melting temperature agarose for injection.
+ Insert needles obliquely into the sinus venosus and inject beads.
+ Reorient an injected embryo in 1% low melting temperature agarose for imaging using confocal microscope.
+ 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.