This video demonstrates a procedure for mounting drosophila embryos to image hemo cytes. Their embryonic macrophages. Embryos are laid by flies on apple juice agar plates in a laying cage overnight.
The next day the agri plate is removed and the embryos are washed from the agri plate to a cell strainer following additional washes to remove debris. The cell strainer containing embryos is submerged in bleach to coate the embryos. After washing off the bleach, the embryos are transferred to a droplet of water.
The droplet of water is then aspirated and the embryos are covered in hello carbon oil. Appropriately staged embryos with visible he cytes are transferred to a Petri per membrane between two affixed cover slips and carefully aligned in oil. A third cover slip is then placed over the embryos and glued into place with nail varnish.
The motility of hemo cytes within the embryo can now be imaged through the uppermost cover slip on a microscope. Hi, I'm Jennifer Zaed from Brian Strm laboratory at the rental division of Seoul and Molecular biophysics at Kings College London. And I'm Ian Evans from the Wood Lab in the Department of Biology and Biochemistry at the University of Bath.
Today we'll show you a procedure for mounting Josephs bryo to live image, the hemo site, the embryonic macrophages of this organism. We use this procedure to study the developmental dispersal and wind response to these important immune cells and the cytoskeletal machinery they utilize to carry out these processes. So Let's get started.
Begin this procedure by obtaining appropriate TROs OAL lines containing hemo cyte specific gal four drivers and genetically encoded fluorescent reporters under UAS control. Here, serpent gal four is used to drive expression of a fluorescent red nuclear marker from UAS Red Stinger. For a discussion of the range of recommended GAL four drivers and UAS constructs, please see the accompanying written protocol place 20 drosophila of each sex in a laying cage.
The laying cage is comprised of a 55 milliliter apple juice agri plate fitted to the bottom of a plastic tube with gauze covering the other end to allow airflow. Alternatively, a simple plastic peaker can be used with holes. Perforating the base, allow the flies to acclimatize to the laying cage for at least two days after the flies have acclimatized change to a new prewarm apple juice grate and allow the flies to lay overnight at 25 degrees Celsius.
For information regarding the collection of more discreetly staged embryos, please see the accompanying written protocol. Once the flies have incubated for the appropriate amount of time for laying, use a wash bottle to apply approximately three milliliters of water to the plate. Then using a soft tipped paintbrush, dislodge embryos from the apple juice, aate dislodged embryos can be easily seen with the naked eye holding a cell strainer over a beaker.
Pour the water from the apple juice aate into the cell strainer to collect the embryos. Then add more water to the apple juice.Aate. Repeat the straining process until the desired number of embryos has been collected.
Next, using a wash bottle, wash the embryos in the cell strainer using approximately 10 milliliters of water. Once the embryos have been washed, place the cell strainer in the lid of the apple juice Aerate. Add neat bleach enough to suspend embryos in the cell strainer to coate the embryos.
Transfer the cell strainer and petri dish lid to a dissection microscope to follow the decoration of the embryos. Under brightfield illumination, decoration is complete when the dorsal appendages have dissolved, which should occur within two minutes. After decoration is complete, remove the cell strainer containing the embryos from the bleach, again, holding the strainer over a beaker.
Use a wash bottle to gently but thoroughly wash off residual bleach. Apply blue colored laboratory tissues to the underside of the cell strainer to blot off the remaining water. If the blue color changes to white or pink residual bleach is present, continue washing ensuring that all traces of bleach have been removed before proceeding to the next step.
Removing excess water from the cell strainer makes it easier to pick up embryos with a paintbrush in the next step. Once all the bleach has been removed from the embryos, place a droplet of water in a Petri dish lid with a fine paintbrush. Collect all the coated embryos from the strainer and resuspend them in the droplet.
Next, apply a chem wipe to the outside of the cell strainer to dry the embryos. Once the embryos have been dried, add a drop of halo carbon oil, 700 to cover all the embryos. Then place a second small drop of oil adjacent to the droplet containing the embryos.
Inspect the embryos under a fluorescent dissection microscope. Identify appropriately staged embryos of the desired genotype to image lateral migration of he cytes. On the ventral midline.
Choose stage 13 to 14 embryos. In this example, he cytes are identified by the fluorescent nuclei and are visible in the head and along the ventral nerve cord and developing dorsal vessel to image the motility of hemos following dispersal over the embryo, choose stage 15 embryos. At this stage, hemos will have dispersed over the entire embryo.
However, three parallel lines of hemos should be visible on the ventral side of the embryo following lateral migration from the midline. Once the embryos have been selected, use a pair of curved number five watchmakers forceps to scoop up the selected embryos. Taking care not to puncture vial membranes.
Then place the embryos in the second drop of oil on the underside of a 50 millimeter Petri perm lumax dish. Place two small drops of Halo Caron oil 700 about one centimeter apart. Apply a cover slip to each drop under brightfield illumination on a dissection microscope.
Carefully transfer up to 15 embryos using curved forceps. Aligning the embryos ventral side up and parallel to the edge of the cover. Slips once the embryos are aligned at a small drop of oil and allow it to spread to form a homogenous layer between the two cover slips.
After the oil has spread, this may take a few minutes. Check that the embryos are still ventral. Side up if the embryos have rolled slightly.
Reposition them again with the forceps. Finally using number three tweezers. Place a third cover slip over the embryos.
Bridging the two previously adhered cover slips. Glue this cover slip to the cover. Slip supports using nail polish.
The embryos are now ready for imaging. This SRP gal four UAS Red Stinger embryo was mounted ventral side up and time-lapse images were acquired on a Leica M 2 0 5 fluorescent dissection microscope. He cytes are visualized by their red labeled nuclei.
The movie begins at stage 12 to 13 of development when the he cytes leave the head and migrate down the ventral midline. After about one hour, he cytes begin migrating off the midline to disperse to lateral positions along the ventral surface. For the remainder of development.
The hemo cytes are highly active, migrating throughout the embryo. We have to show you how to monitor Joseph embryo to follow the movement or the hemo side live in vivo. When doing this procedure, it's important to handle the embryos carefully, especially on the petro perm dish, to avoid damaging either once the embryos are in oil, they're relatively resistant to dehydration, but care should be taken not to bleach the embryos for too long when removing the corion.
Lastly, by mounting several embryos, you will give yourself the best possible chance to obtain an embryo in the perfect orientation for live imaging. So that's it. Thanks for watching and good luck with your experiments.
