The overall goal of this procedure is to follow the dynamics of apoptotic cell clearance in the drosophila embryo. This is accomplished with transgenic flies containing a cytoplasmic GFP marker, which labels phagocytic cell populations embryos with the Simio GFP marker are collected, washed, and coated. Next, the embryos are positioned for injection followed by monitoring of the CNS.
The third step is the labeling of apoptotic cells by injecting the embryos with specific apoptosis phagocytosis markers, such as an X in five, the final step is to make time-lapse recordings of the embryonic CNS using confocal microscopy. Ultimately, the results can show the localization and behavior of apoptotic cells and phagocytes during different stages of apoptotic cell clearance through fluorescence confocal microscopy. The main advantage of this technique over existing methods like immunohistochemistry on fixed embryos is that we can follow dynamics of apoptotic cell clearance.
Apoptotic cell clearance is a highly dynamic process containing diverse steps. This technique allows us to follow the different steps using specific markers for apoptotic cells and phagocytes. The protocol begins with making a glue from conventional reagents unroll double-sided tape, then roll it back up to fit into a scintillation vial.
Fill the vial with heptane and seal it with param. Then attach the vial to a nut tater and gently rock it for 24 hours. At room temperature, the next day the prepared glue is transferred to a new vial.
Use a glass PEs your pipette to transfer the prepared glue to a new file. Once prepared, use a pipette tip to apply the glue to cover slips. Disperse a drop of heptane glue into a line down the middle of the cover slip.
Let the glue try for a few seconds. The cover slips can then be stored at room temperature in a closed box for up to a month after anesthetizing the flies with carbon dioxide Pool about 400 flies in a collection container with a warmed grape juice laying plate replaced in 25 degrees Celsius for two hours. Collect the grape juice laying plate and cover the container with a new plate to keep flies happy.
The collected plate contains zero to two hours old embryos. Return the collected grape juice laying plate with the embryos to a 25 degrees Celsius incubator until they reach the desired developmental time point. In this example, embryos are incubated between 10 and 12 hours, so they develop to stage 15 or 16 after they've developed.
Use tap water and a clean paintbrush to transfer the embryos from the plate to a cell.Strainer. Keep the strainer over a container so that any embryos that spill can be recovered. Rinse the collected embryos until all of the yeast paste is removed.
Then dry out the excess water by wiping the outside of the strainer. Place the cell strainer in a clean Petri dish to remove the corion at enough. 50%bleach to cover the embryos in the cell strainer and wait two minutes, stirring them two to three times.
Next, rinse off the embryos until they no longer smell like bleach. Place the embryos in the cell container into a clean Petri dish and cover them with water to prevent them from drying up. For injecting the embryos.
Prepare injection needles in advance using a needle puller and thin walled glass filaments. Load one microliter of the desired reagent into two needles with one needle serving as a backup. A stable marker that does not bleach easily, such as an X in five makes a good reagent.
Now place a rectangular piece of grape juice agar on a microscope. Slide and transfer the embryos from the strainer to the agar. Using a clean paint brush under a fluorescent dissection microscope, select properly staged embryos for injection using the GFP marker.
Using a wet paintbrush, move the selected embryos into a row of 10 with their ventral sides up. Position the row at the edge of the agro block. GFP can be seen in the embryo central nervous system.
Now attach the embryos to a cover slip with a strip of the heptane glue. The right position of the embryos is very important for the success of the injection. Take your time and make sure the embryos are lined up precisely in the proper orientation.
Next, place the cover slip in the dehydration chamber for about five minutes. After drying the embryos, cover them with Hello carbon oil 700 to avoid further dehydration. Lastly, place a drop of water on a microscope slide.
Then put the cover lip with the embryos up on the wet microscope slide. The embryos are now ready for injection. Begin by attaching a needle to a microm manipulator and the pico pump.
The pump controls the amount of injected liquid by pushing nitrogen into the needle. A foot pedal controls the gated ejection of the nitrogen under the microscope. Focus on the needle tip and position it near the edge of the cover slip with the needle tip and the cover slip in focus very carefully.
Move the cover slip until it hits the needle tip and breaks it. The opening diameter should be one half to two micrometers. Now focus on the embryos.
Bring the needle into the oil. If the needle tip has been broken correctly, there should be a drop of liquid leaking into the oil. When you press the pedal without touching the needle holder, move the stage so that the lateral edge of the embryo is pierced by the needle.
Quickly inject a drop of solution into the embryo. Move the embryo away and proceed to the next one until all 10 of the embryos are injected. Image the embryos on an inverted confocal microscope with a 40 x or 100 x objective.
Search for a nicely positioned embryo with the CNS in the middle, showing strong GFP expression and labeling of apoptotic cells. To make time-lapse recordings, choose five or six confocal slices to micron stick at these locations. Take a scan every 60 seconds an interval at which GFP bleaching should not be an issue.
Afterwards, make a projection of three slices with a six micron thickness to observe the whole cells. In some cases, an embryo may start rolling during the time of video recording. Therefore, monitor the recording so no time is lost on moving embryos.
A stage 15 embryo properly positioned shows the embryonic CNS in the middle with well labeled glia marked by a g macrophages M, which are mostly outside the CNS show. Strong cytoplasmic GFP expression. Ectodermal cells E are also labeled with cytoplasmic GFP.
These frames show apoptotic cells labeled with an ex in five and clea ectoderm and macrophages labeled with cite GFP. Each frame is a projection of three slices and each slice is two microns thick. Many an ex in five positive cells are inside and outside the CNS.
The boxed event shows a glial cell engulfing an apoptotic particle. Note the probing behavior of the glial cell. It touches the apoptotic particle a few times before committing to engulfment of the apoptotic cell After its development.
The technique paved the way for researchers in the field of apoptotic cell clearance to explore phagocytosis of apoptotic cells during development. Using this technique, we can explore the highly dynamic behavior of apoptotic cell clearance.
