This video explains how to perform transmission electron microscopy, also known as TEM on the drosophila embryonic cart. The heart has emerged as a valuable model system for studying cell migration, cell cell adhesion and cell shape changes. This technique allows visualization of heart lumen shape, as well as ultra structural components such as cell membranes, cell cell junctions, and basement membrane.
To begin this procedure, a fixation solution appropriate for transmission electron microscopy of drosophila embryos is prepared. Next, a zero to 20 hour collection of coated embryos are placed into the fixation solution. When finished, embryos are removed from the fixation solution and handy ized while attached to double-sided tape.
In a Petri dish containing buffer deionized embryos float in the buffer and are recovered with a pipette. Then embryos are postfix and undergo a dehydration gradient. Once infiltrated with resin, they're transferred to an embedding mold where they're aligned.
The resulting block is cut with a diamond knife. Sections are picked up on copper grids, stained and examined. Hi, I'm Nadine Lop from the laboratory of Sunita Kramer in the Department of Pathology and Laboratory Medicine at the University of Medicine and Dentistry of New Jersey at Robert Wood Johnson Medical School.
I'm Raj Patel. I'm the electron microscopist also from the PON pathology. Today we will show you a procedure for preparing and imaging oph embryos for transmission electron microscopy.
We use this procedure in our laboratory to study Drosophila heart morphogenesis. So let's Get started. Get started.
Before handling the drosophila embryos freshly prepare fixative solution for the Heptane permeation method of embryo fixation. In a fume hood pipette two milliliters of a freshly made solution containing 12.5%glutaraldehyde in Cate buffer into a glass scintillation vial. Add eight milliliters of ADA heptane and shake vigorously.
Then wait one minute. Allowing the two phases to separate the upper phase contains the ADA heptane, saturated with glutaraldehyde, which is the fixative solution. Pipette the upper phase into a clean vial and set it aside To collect zero to 20 hour embryos, use an embryo collection chamber, which is a small basket with a removable mesh insert.
Begin by removing the grape juice plate containing the embryos and remove any dead flies. Then using water and a paintbrush. Loosen embryos from the grape juice plate, transfer embryos into the embryo collection chamber using a squar bottle containing water.
Rinse the embryos of any excess yeast from the grape juice plate. Remove the outer corion membrane by soaking embryos in a 50%bleach solution for two to three minutes or until embryos float to the surface. Rinse thoroughly with distilled water and flo the embryos on a paper towel.
Using forceps, pick up the mesh insert, which should now be covered with coated embryos. Place the insert into the fixative, allowing the embryos to fall from the mesh. Allow embryos to fix for one and a half hours at four degrees Celsius while mixing on an orbital rocker.
Once the incubation is finished, place the embryos into a Petri dish lined with double-sided tape taking as little of the fixative as possible. Shake the dish to spread the embryos into a single layer, and then place it in the chemical hood to allow the heptane to evaporate. Once the embryos have dried, cover the embryos with buffer containing 0.1%tween hand de vize.
Late stage 16 embryos under a dissecting microscope with a blunt tungsten needle. Embryos that have been ized, float and can be recovered with a pasture pipette transfer embryos to a 1.5 milliliter micro fuge tube. Rinse embryos in 0.1 molar cate buffer, and then postfix in 1%Osmium tetroxide for one hour at room temperature.
Following the incubation, rinse the embryos with Cate buffer. Once again, dehydrate embryos in a grade series of ethanol and acetone. Start with 50%ethanol for 10 minutes, followed by 70%Ethanol for 10 minutes, then dehydrate in 90%acetone for 10 minutes, and finally into 100%acetone steps of 10 minutes each.
Remove the acetone. Begin the infiltration process by adding a one-to-one mixture of ein spray resin and acetone. Ensure that the sample is under vacuum pressure and microwave for three minutes.
Maintain vacuum pressure for an additional five minutes. Exchange the resin acetone mixture with 100%resin and microwave again under vacuum pressure. Exchange the resin and microwave under pressure.
A final time for a total of three infiltration steps. Transfer the embryos from the micro fuge tube to a silicone embedding mold. Align the embryos into a row such that the posterior end of the embryo aligns with the tapered edge of the mold.
Bacon is 70 degrees Celsius oven overnight. Then remove the resulting block for sectioning. Use a microtome to cut one micrometer sections into the embryos.
Once the block has been sectioned through and the embryo has been reached, use a loop to move a single section onto a glass slide to determine whether the proper depth for visualizing the heart has been achieved prior to visualization. Briefly heat the section on a hot plate and stain the section with methylene blue by first adding a drop to the slide. And then briefly heating.
Again, rinse with deionized water and examine the section under a microscope at the desired depth of sectioning. The heart lumen should be recognizable with a light microscope to satisfy space constraints. Re trim the block to include a maximum of five embryos in cross section that show the embryonic heart.
Cut 90 nanometer sections using the microtome. Pick up multiple sections on a copper grid. Stain the grid with 3%al acetate for 10 minutes, then rinse three times with distilled water.
Now stain the grid with lead citrate for two and a half minutes in a chamber containing sodium hydroxide pellets to create a carbon dioxide free environment. After the incubation, rinse three times with distilled water. Examine the sections with an electron microscope at 80 kilovolts and photograph with a CCD camera.
In order to determine whether the sectioning depth and staging were properly performed, it should be apparent that a mature heart lumen has formed between the two cardio blasts of the dorsal midline to determine whether cell membranes are intact, examine the heart lumen for the presence of extracellular matrix and look for the presence of Christie and mitochondria. So we've just shown you how to determine the ultra structure of the drosophila embryonic heart. When doing this procedure.
It's important to remember to properly stage the drosophila embryos and to accurately measure the components of the resin and mix them well. So thanks for watching and good luck with your experiment.
