CHAPTER 4 - MICROSCOPIC OBSERVATIONS
AGAR MOUNTING
(Source: J. Eisen)
For more precise orientation of zebrafish embryos and to keep them from floating around during long term observations, embed them in agar.
Mounting
1. Prepare a 1.2% solution of agar in embryo medium (or Ringers solution if the embryos are to be injected or prepared for some other invasive procedure; see RECIPES, Chapter 10) by heating in a boiling water bath or in a microwave oven. This solution remains liquid at 37-40°C, but quickly hardens when cooled.
2. Keep a small amount of solution (5 ml) liquid in a test tube in a 42°C water bath.
3. Keep an extra glass plate from the stage of the stereomicroscope warm by immersing it in the water bath.
4. Just before mounting the embryos, replace the room temperature glass plate with the warm one; be sure to dry it completely before using it.
5. Take the test tube containing the agar out of the water bath and transfer an embryo in a minimum amount of Ringers solution into it.
6. Place a viewing chamber (1) on the warm glass plate on the stereomicroscope. Quickly suck up the embryo in a small drop of agar and transfer it onto the chamber, keeping it clear of the cover slips that have been glued on.
7. Using a fine minutien pin (0.10 mm diameter) mounted on a needle holder or on an applicator stick, position the embryo at the appropriate angle in the agar and allow the agar to harden.
8. After the agar hardens, use a razor blade to cut out a small block of agar containing the embryo. Slide the block, in the appropriate orientation, until it is pushed against the glued-on cover slips. Remove residual liquid surrounding the agar block with a Kimwipe. Place a small drop of liquid agar over the agar block to hold it in place. When this hardens, cover the preparation with Ringers solution, as with embryo medium.
Removing embryos from agar
1. To remove an embryo from the agar, place several drops of medium on top of the agar.
2. Using a pointed scalpel or a minutien pin attached to a needle holder or applicator stick, create a "V" shaped cut in the agar with the point of the "V" aiming towards the embryo's head.
3. Hold the tips of a pair of sharp forceps closed and plunge them into the agar between the point of the "V" cut and the head of the embryo.
4. Gently open the points of the forceps creating a crack in the agar which will run along the length of the embryo.
5. The embryo will then float out of the agar into the saline and can be picked up with a fire-polished Pasteur pipette.
The Zebrafish Book
