Layered Agar Mounting: Preparing Live Zebrafish Embryos for Long-Term Imaging with an Inverted Microscope

- Start with a Petri dish containing embryos in E3 medium. Add tricaine to anesthetize the embryos and phenylthiouria, PTU solution, to inhibit pigment formation. With the help of a dissecting microscope, dechorionate the embryos.

Use a glass pipette to transfer one of the dechorionated embryos to a small Petri dish with a shallow glass bottom well in the center and remove the excess medium. Next, place an agarose solution at an optimum concentration to minimize embryo distortion and motility into a microfuge tube and heat it. Bring down the temperature to 30 degrees Celsius to avoid damaging the embryo due to heat.

Pour the first layer of agarose over the embryo, completely filling the shallow well. Place another cover glass over the well and add 1% agarose on top of that. The second layer holds the cover glass in place.

Once the agarose solidifies, fill the Petri dish with the E3 medium containing tricaine. This is the third layer and keeps the agarose and embryo hydrated. In the following protocol, we will perform layered agar mounting of zebrafish embryos for extended time lapse imaging.

- Just before mounting, heat the agarose solution to 65 degrees Celsius and then let it cool to approximately 30 degrees Celsius so the embryo is not harmed by the heat and add tricaine to the agarose solution. Use 35 millimeter glass bottom dishes with a number zero covered glass bottom. Gently place a dechorionated embryo with one of its lateral sides toward the bottom of the dish with a glass pipette or a micropipette. Then carefully remove any remaining E3 with a micropipette.

Add the first agarose solution to the small well created by the cover glass attached to the bottom of the dish to cover the embryo, making sure that the agarose covers the small well but does not overflow it. Cover the small well with the cover glass to create a narrow agarose-filled space with the embryo between the two cover glasses. Place a layer of 1% agarose solution on top of the cover glass all over the bottom of the dish, which will keep the cover glass in place as it solidifies. Then fill the remaining portion of the dish with E3 containing 0.02% tricaine to keep the system hydrated. To identify the optimal agarose concentration for layer one, mount the embryos in increasing concentrations of agarose and perform timelapse imaging to identify the concentration that minimizes embryo distortion and motility, then test a finer range of concentrations based on the results.

- The most critical step of this protocol is to identify the optimal concentration of agarose for layer one. Test different nano concentrations of agarose such as 0.030%, 0.032%, et cetera, to find the optimal concentration.