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*4.5.3 Dye injection

Misato Fujita~
Tokyo Institute of Technology

**4.5.3.1 General information

Confocal microangiography method has difficulties in imaging deep or complicated areas. These limitations are compensated by the berlin-blue dye (Wako) injected embryos (Ura, 1944). 

**4.5.3.2 Preparation

+ Prepare 0.75 % berlin-blue dye solution in deionized distilled water.
+ Prepare the grass microinjection needles by grass capillaries using a vertical pipette puller.

**4.5.3.3 Dye injection

+ Dechorionate the required staged embryos by manipulation.
+ Anesthetize the dechorionated embryo with 0.84% tricaine (Sigma).
+ Tear the yolk for ease of injection and incise the heart to allow blood drainage.
+ Inject the berin-blue dye with the grass microinjection needle from the dorsal aorta.
+ Fix the injected embryo in 4% paraformaldehyde.
+ The specimens are observed through the stereomicroscope.

**4.5.3.4 Reference

Ura R (1944). The development of the blood vascular systems. Nisshin Igaku 33:130-143.