Separating the major organelles inside fruit fly eggs by density starts with harvesting embryos, which are less than three hours old. The eggshells are then removed by incubating in 50%bleach after decoration. Embryos have defined stages are mounted in agar.
As a final step, the embryos are centrifuge and recovered, examining the recovered embryos using bright light and fluorescence. Microscopy shows the localization of specific proteins to certain organelles. A major strategy for purifying and isolating intracellular organelles is to separate them from all other cell components by density, but there's a challenge and cells are disrupted prior to centrifugation proteins and organelles that are usually separate.
Mays stick to each other under these non-native in vitro conditions. Conversely, some proteins might fall off structures they usually bind to. The main advantage of the technique we are going to describe is that it allows separation by density in vivo.
Thus, it avoids breaking open cells and the artifacts that come with it. This method can help answer key cell biological questions. It makes colocalization experiments very easy and allows the isolation specific organal fractions for transplantation experiments and for biochemical analysis.
In this procedure, high percentage agar medium containing apple juice is prepared and poured into plates as detailed in the accompanying written protocol. The plates are used both at the egg laying step and for the subsequent embedding and centrifugation of the embryos. This procedure begins with initiating egg collection.
First, prepare yeast paste by mixing dry yeast with water to peanut butter. Consistency then cover a portion of a fresh apple juice agar plate with the yeast paste. Adult flies are kept in cages to which agar plates are affixed at the bottom.
To remove the current plate, invert the fly cage and bang it on the bench top. A few times the flies fall to the bottom and are briefly disoriented in this very short period. Quickly replace the old agar plate with the fresh plate layered with the yeast paste.
The yeast provides nutrition for egg production, and together with the apple juice in the agar, it induces egg layering. Discard the first 30 to 60 minutes egg collection. It yields a large fraction of misstaged embryos since they were held by the females after fertilization.
Subsequent egg collections tend to be dominated by eggs deposited shortly after fertilization. Collect eggs for up to three hours since most consistent layering is achieved in young embryos. Shorter collection times of one hour or less are preferred.
For routine experiments, use tweezers to remove any flies stuck to the yeast or to the surface of the agar plate. After collecting the eggs, proceed to remove the egg, shell place the agar plate under a dissecting microscope and observe the embryos by transillumination. Make sure to identify the egg filaments to remove the eggshell from the embryos.
Squirt 50%bleach under the agar plate, covering the embryos and incubate for several minutes. Agitate the agar plate occasionally to swirl the embryos around in the bleach. Examine the eggs again under the microscope.
Once the egg filaments are no longer visible, the Corian has been removed successfully. This typically requires three to five minutes of incubation with the bleach solution. Next, use a wire mesh basket as a sieve to separate the embryos from the bleach.
The basket is prepared from a two centimeter by two centimeter square of a wire mesh flat sheet indented in the middle. Grab the wire basket with tweezers and hold it over the inverted cover of the agar plate. Pour the bleach and embryo slurry from the agar plate through the wire mesh draining into the agar plate lid.
If embryos remain on the agar plate, squirt distilled water onto the plate to dislodge them and pour the distilled water and embryo slurry through the wire mesh. Touch the bottom of the wire mesh to a paper towel to bloss away any excess liquid. Then squirt distilled water along the perimeter of the wire basket to rinse off the remaining bleach while minimizing embryo loss.
Frequently blot off excess liquid. Repeat the washing step five to 10 times until all bleach has been removed. If the wire basket still smells of bleach, continue to wash the embryos.
Once the embryos are sufficiently washed, they can be mounted in agar. Wash the embryos outta the wire basket. With TSS into an empty petri dish, the embryo should sink to the bottom.
On a dissecting scope, observe the embryos by trans illumination. Specific stages can easily be recognized visually. Using a P 200 pipette select embryos of desired stages and transfer them to a small agar plate.
Work quickly since the embryos are aging and prolong submersion of longer than 30 minutes, in TSS may affect developments using a pipette. Under Kim wipe. Remove most of the TSS.
The surface of the agar should be slightly moist, which makes it easier to push embryos around. However, there should not be pools of liquid remaining anywhere on the plate since excess liquid during centrifugation will cause the embryos to slide out of the holes. The single most difficult aspect of this procedure is mounting the embryos and auger.
This is particularly challenging because the embryos are delicate and can easily be destroyed and handled to roughly. To prevent this, the needle must be moved slowly, deliberately, and in small increments. Practice is the best way to learn exactly how much touch and pressure the embryo can withstand Mount a tongues and needle backwards in a needle holder so that the blunt end sticks out while observing.
Under the microscope, use the needle to poke vertical holes in the agar near each embryo to be able to simultaneously watch under the microscope while poking the hole into the agar, the needle is held at an angle. It is therefore hard to poke holes at a perfectly vertical. However, the minor inclination of the needle creates a slight depression or groove on one side of the hole so that the embryo easily slides along it and into the hole.
Next, identify the anterior and posterior ends of the elongated embryos as the embryo should be pushed into the holes in a consistent orientation. Typically with the anterior end up, the ign membrane at the anterior end is characterized by a specialized structure. The micro pile, using the blunt end of the needle, push the embryo to move it along the agar and to orient and maneuver its posterior end towards a punctured hole.
Then push the anterior end towards the hole. The embryo should slide easily into the hole along the slight depression generated during puncturing. As the embryo tilts up, push it deeper down into the hole.
The agar is soft enough that once an embryo is partially inserted into a poked hole, it can be pushed in further without damage. When fully pushed in, the embryo is usually already fairly vertical. If not align the inserted embryo more vertically, we're pushing on it sideways.
If the hole is not too large, the embryo position will be stably held by the agar throughout centrifugation. With practice, it is possible to embed 100 to 250 embryos per plate. The number of embryos required depends on the downstream application.
Keep the mounting time short. If a narrow developmental stage is desired since the embryos continue to develop during mounting, remove leftover unused embryos with a moist and fly brush. Next, the embryos mounted in agar a centrifuge.
Transfer the plates holding the fly embryos to the swinging buckets of the centrifuge agar side down. Make sure the centrifuge is evenly balanced and centrifuge the plates at 4, 000 rotations per minute for 30 minutes at four to 10 degrees Celsius. After centrifugation, place the agar plate under the dissecting microscope and cover it with a layer of TSS using the blunt end of the needle.
Dig the embryos out of their holes. The embryos float in the TSS but remain submerged. The embryos appear obviously layered with a brown cap at the anterior end, a clear middle region and a dark gray area at the posterior end.
Discard embryos that fail to show distinct layering for direct observation by brightfield or epi fluorescence microscopy. Use a P 200 pipette to transfer well layered embryos in TSS to a micro centrifuge tube. Then transfer the embryos in buffer to a glass slide, a mount as described in the accompanying written protocol.
If the embryos are to be fixed, use a P 200 pipette to transfer well layered embryos in TSS to a scintillation vial. Fix the embryos using standard fixation techniques. Since for centrifuge embryos, the efficiency of the devitalization step is low.
Start with as many embryos as is practical. If embryos are to be used in transplantation experiments, transfer the embryos to agar plates and mount on cover slips. Using standard embryo handling procedures, references to such procedures can be found in the written protocol.
The layering induced by centrifugation is stable for tens of minutes. Here are bright light images of living oph embryos prior to centrifugation shown with anterior end to the left and posterior end to the right dorsal side. Up and ventral side down cleavage stage embryos appear uniformly.
Brown stages like this are ideal for in vivo centrifugation. Blaster derm embryos have a clear rim that appears similar on all sides. This clear periphery expands until near the end of ization.
Embryos can still be successfully stratified by centrifugation until the end of ization. The layering of yoke and nuclei is not as consistent as in cleavage stages. After cellular embryos appear asymmetric, the dorsal and ventral ventrals become distinct and various folds develop.
The embryo shown is in early germ band extension. In these stages, centrifugation is no longer effective at stratifying the major organelles. Embryogenesis is a continuum.
Therefore, these stages are not discrete entities, but they turn into each other as development proceeds. This time lapse movie captures four hours of embryo genesis from cleavage stages. The germ band extension, it is sped up 720 fold.
Here are images of centrifuge embryos taken using trans illumination. The embryo on the left stratified well. The low density lipid droplets form a lipid cap at the very anterior tip.
The high density yolk vehicles accumulate as a dark gray zone at the posterior end. These two layers are separated by a broad clear that represents other organelles and cytoplasm. The embryo on the right was centrifuged after ization.
Layering is minimal if an epi fluorescence microscope is available. These living centrifuged embryos can be examined under UV excitation where the yolk displays intense blue water fluorescence. A compact layer of autofluorescent material at the posterior pole indicates successful centrifugation and serves as a marker for the posterior end.
The appearance of the layers changes drastically if the embryos are fixed. Lipid droplets become translucent and the lipid layer appears whiteish and fluffy by bright light microscopy rather than dark brown. Yo corso fluorescence is partially or completely destroyed by fixation becoming much less intense or even undetectable.
In this image, organelles are labeled with fluorescent markers and the markers accumulate characteristic positions along the anterior posterior axis. Certain histone fusion proteins localized to both lipid droplets and nuclei, and thus can be used to mark these cellular compartments as well as to monitor the stage of the embryo by the number of labeled nuclei. As you've just seen, one of the most challenging aspects of this procedure is embedding the embryos into the auger.
But with some practice, you'll be able to do this quickly and efficiently. Once mastered. This technique makes otherwise tedious colocalization experiments very easy.
For example, our laboratory routinely uses this technique to test by fluorescence microscopy if a protein of interest localizes to a specific major organelle. In addition, individual layers can be recovered from the centrifuge embryos for biochemical analysis or transplantation experiments. This technique is not only useful for fruit fly embryos.
Centrifugation also separates organelles by density in the eggs of other species and in other large cell types such as cytes.
