Encyclopedia of Experiments: Biology
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Transcript
- 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 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 to 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 to the very bottom of the pipette. Then transfer the embryos to 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 to gently mix the embryos in the agarose.
Then, with the pipette, transfer the agarose and embryos to the well of a glass bottom six-well plate. Under a stereo microscope, use a gel loading tip fixed to the end of a teasing needle to position the embryos close to the cover glass and in the desired orientation for imaging. After the agarose has set and medium with tricaine has been added to the wells, use an inverted wide-field epifluorescence laser-scanning confocal or spinning disk confocal microscope to acquire images.
For a whole embryo field of view, use a 4x subjective. Use a 20x objective to image a specific tissue. Process the images according to the text protocol.
