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

1. Microscope Setup, Image Acquisition, Processing and Analysis

1. Prepare 0.8% low melting point (LMP) agarose: Add 0.8 g LMP agarose to 100 ml E3 and heat until fully dissolved. While hot, aliquot 1 ml of LMP agarose into 1.5 ml microfuge tubes in a 37 °C heating block. The LMP agarose will remain molten at 37 °C. Add 40 µl of 4 mg/ml (25x), pH 7.0 tricaine to each 1 ml aliquot of LMP agarose at 37 °C. NOTE: If working with a pigmented strain, add 20 µl of 0.15% (50x) PTU to each 1ml aliquot.
   1. Store the remaining LMP agarose for several months in sealed 50 ml tubes at 4 °C.
2. Anesthetize parabiotic embryos to be imaged by adding 1 ml of 4 mg/ml (25x), pH 7.0 tricaine to the 25 ml of E3 that the embryos are already in.
3. Gently swirl the dish so that the embryos will pool in the middle. Then, using a wide-tipped plastic transfer pipette, draw up the embryos in as little liquid as possible. Turn the pipette upright and gently bounce the embryos so that they settle to the very bottom of the pipette.
   1. With the embryos at the bottom of the pipette, transfer them to a 1 ml aliquot of LMP agarose by lightly touching the pipette tip to the surface of the agarose. Avoid transferring excess liquid as this will dilute the agarose.
4. After expelling the excess liquid from the pipette, use the pipette to gently mix the embryos within the agarose, then use the pipette to transfer all of the agarose and embryos to the well of a glass-bottom (No. 1.5 coverslip), 6-well plate.
   NOTE: Be sure to discard the pipette after transferring the embryos to the imaging plate. Residual agarose will set inside the pipette, rendering it ineffective for further use. Rather, use a new pipette each time.
5. Under a stereoscope, use a gel-loading pipette tip fixed on the end of a wood-handled teasing needle to position the embryos down towards the cover glass (to ensure they are within the working distance of the microscope objective) and in the desired orientation for imaging.
   NOTE: Reposition continuously until the agarose has fully set. Take care to mount the parabiotic embryos according to which embryo of the pair is to be imaged. Given the random orientation of the conjoined embryos, for some pairs it may be difficult to image both embryos. In this scenario, recover the embryos from the agarose using forceps and a wide-tipped plastic transfer pipette and then remount for imaging from a different perspective.
6. Once the agarose has set, cover with 2 – 3 ml of E3 supplemented with tricaine (and PTU if necessary).
7. Image the embryos using an inverted wide-field epi-fluorescence, laser-scanning confocal, or spinning disk confocal microscope. Select the objective most appropriate for the desired imaging. For a whole-embryo field of view, use a 4x objective. Select a higher magnification, such as a 20x objective, to image a specific tissue
   NOTE: If using an upright microscope, mount in LMP agarose (similar to above) on a glass cover slip. Invert the glass coverslip onto a glass microscope slide that has a ring of vacuum grease filled with the same E3-tricaine-PTU.
8. Process acquired images using free image analysis software packages such as NIH Image J/FIJI.
   NOTE: Count cell numbers and quantify dynamics such as cell migration and cell division using segmentation and tracking algorithms.