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

– Begin by adding E3 medium 세스 low melting point agarose and heating until the agarose completely dissolves. Cool the agarose 세스 37 degrees Celsius and add tricaine solution. Next, anesthetize the embryos by adding tricaine 세스 a Petri dish containing embryos in E3 medium. Swirl the Petri dish 세스 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 세스 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 세스 position the embryo in the desired orientation. Once the agarose solidifies, pour E3 medium containing tricaine on top of it 세스 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 in a 37 degrees Celsius heating block. Anesthetize the parabiotic embryos by adding 1 milliliter of 4 milligrams per milliliter pH 7.0 tricaine 세스 the 25 milliliters of E3 medium containing the embryos. Gently swirl the dish so that the embryos pool in the middle.

Then using a wide-tipped plastic transfer pipette, draw up the embryos in as little liquid as possible. Next, turn the pipette upright and gently bounce the embryos so that they settle 세스 the very bottom of the pipette. Then transfer the embryos 세스 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 세스 gently mix the embryos in the agarose.

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

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