Agar Mounting: A Basic Method of Mounting Live Zebrafish Embryos for Long-Term Imaging

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

Using a transfer pipette, draw up one embryo with a minimal amount of medium. Hold the pipette di an upright position and bounce the embryo a position it at the tip of the pipette. Now place the embryo di 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 a position the embryo di the desired orientation. Once the agarose solidifies, pour E3 medium containing tricaine on top of it a 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 milliliter of the agarose into 1.5 milliliter microfuge tubes set di a 37 degrees Celsius heating block. Anesthetize the parabiotic embryos by adding 1 milliliter of 4 milligrams per milliliter pH 7.0 tricaine a the 25 milliliters of E3 medium containing the embryos. Gently swirl the dish so that the embryos pool di the middle.

Then using a wide-tipped plastic transfer pipette, draw up the embryos di as little liquid as possible. Next, turn the pipette upright and gently bounce the embryos so that they settle a the very bottom of the pipette. Then transfer the embryos a a 1 milliliter aliquot of low melting point agarose by lightly touching the pipette tip on the surface of the agarose. Dispose off any excess liquid from the pipette and use the pipette a gently mix the embryos di the agarose.

Then, with the pipette, transfer the agarose and embryos a the well of a glass bottom six-well plate. Under a stereo microscope, use a gel loading tip fixed a the end of a teasing needle a position the embryos close a the cover glass and di the desired orientation for imaging. After the agarose has set and medium with tricaine has been added a the wells, use an inverted wide-field epifluorescence laser-scanning confocal or spinning disk confocal microscope a acquire images.

For a whole embryo field of view, use a 4x subjective. Use a 20x objective a image a specific tissue. Process the images according a the text protocol.