Agar-montage: een basismethode voor het monteren van levende zebravisembryo's voor langdurige beeldvorming

- Begin by adding E3 medium to low melting point agarose and heating until the agarose completely dissolves. Cool the agarose to 37 degrees Celsius and add tricaine solution. Next, anesthetize the embryos by adding tricaine to a Petri dish containing embryos in E3 medium. Swirl the Petri dish to collect the embryos in the middle.

Using a transfer pipette, draw up one embryo with a minimal amount of medium. Hold the pipette in an upright position and bounce the embryo to position it at the tip of the pipette. Now place the embryo in the agarose solution without adding any excess medium, which would dilute the agarose and prevent gelling. Gently mix the embryo within the agarose and transfer the solution into the well of an imaging plate using the pipette.

Place the plate under a microscope and use a pipette tip to position the embryo in the desired orientation. Once the agarose solidifies, pour E3 medium containing tricaine on top of it to keep the agar hydrated and image the embryo. In an example protocol, we will mount parabiotic zebrafish embryos for imaging and analysis.

To acquire images of the fused embryos, prepare 0.8% low melting point agarose. While still hot, aliquot one