4.5.2 Confocal microangiography
Tokyo Institute of Technology
220.127.116.11 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).
- 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.
- 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.
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.