The overall goal of this procedure is to label single neurons in the central nervous system of early stage 17 drosophila embryos. This is accomplished by collecting eggs and allowing them to develop to the right stage. The second step of the procedure is to manually chlorinate and align the eggs.
Following this diva, colonization and fileting of the embryos makes the nerve cord accessible. The final step is to label single cells by Iono retic application of the lipophilic die die eye. Ultimately, results allow the visualization of single neuron morphology if desired in the context of known GFP expression patterns and or in mutant background.
The main advantage of this technique over existing single cell labeling methods like markum is that it works in the embryo and allows to visualize identified cells Intentionally. Visual demonstration of this method is extremely helpful as many steps are delicate and therefore difficult to learn. Allow healthy flies to lay embryos on agar plates.
Replace the plate every one and a half hours at 25 degrees Celsius and retain the embryos. Incubate the embryos on their plates at 25 or 18 degrees Celsius until they reach the required developmental stage. When ready, transfer the embryos to a slide covered with double-sided sticky tape.
Avoid transferring any agar with the embryos as it will interfere with their adhesion to the tape. On the tape. Allow the embryos to dry for five to 10 minutes while waiting.
Coat a 24 millimeter by 60 millimeter cover slip with heptane glue. Place a small drop of glue in the center of the slide and spread it into a very thin film using an 18 millimeter square cover slip. Once the embryos have dried, gently touch them with a needle.
The corion will split open and the embryo will stick to the needle. Immediately transfer the dechlorinated embryos to an agar block to prevent them from drying. Align them there 10 in a row, ventral sides up.
Cut away the excess agar from the block with a scalpel. Then gently touch the embryos with the heptane glue coated cover slip. They should adhere to the cover slip with their ventral sides down.
Apply a frame of adhesive tape around the cover slip and attach it to a microscope slide. Keeping the embryos on top of the cover slip. Cover the embryos with generous amounts of PBS on the dorsal side of each embryo near its posterior end.
Penetrate the vitel membrane with a glass needle tear open the membrane along the dorsal midline and drag the embryo out of the membrane. Now reorient the embryo ventral side down elsewhere on the heptane glue substrate. File the embryo with a glass needle by gently perforating the body wall and tearing along the dorsal midline.
Using the needle, push down on the body wall so that it sticks to the slide. Tear through the gut at its anterior and posterior ends, and lift the gut away. Since several embryos will be file on the same slide, it is helpful to either be very accurate with their orientation or make a drawing of their orientation.
Next, the embryos may be lightly fixed for 10 to 15 minutes in 7.4%formaldehyde in PBS followed by four washes in PBS use extreme care to avoid touching the embryos to the surface of the solution. The surface tension forces will destroy them under a 10 x objective. Center the field of view on a prepared embryo, increase the magnification and locate the cell of interest under DIC optics or under fluorescence.
If the target cell expresses GFP, ensure that the field diaphragm of the microscope is opened just to the edge of the field of view, not beyond a narrow illumination. Beam will aid positioning of the micro pipette change back to the 10 x objective and place the bath electrode for the DC amplifier into the solution far from the embryo and the path of the micro pipette. Mount the injection micro pipet filled with lithium chloride solution to the holder and attach it to the micro manipulator.
Using the course controls position the micropipet below the objective and well above the embryo. If the angle of the micro peppe is too steep, the tip will not get in focus. If it is too shallow, the shaft will foul against the bath wall center the tip in the field of view.
Now lower the micro pipette and focus alternately until the pipette is in the PBS and not too far above the embryo. Now change to high power objective and center the pipette within the field of view. When the shaft is in focus, move the micro pipette in the X axis until its tip lies in the center of the field of view.
The micro pipette tip should be well above the level of the embryo. Switch to fluorescence illumination and examine the tip. Check for leakage of dye eye.
Adjust the DC current so that dye eye crystal does not build up at the tip. Now lower the micro pipet in the Z axis towards the embryo. Progressively adjusting the focus as the embryo comes into focus.
Switch to using the fine control. Focus the cell to be injected at the center of the field and then refocus on the micro pipette tip. From this point onwards, it might be more convenient to use the stage control of the microscope to move the embryo relative to the micro pipette.
Bring the micro pipet tip into contact with the cell and make a depression on its surface. Pass several nano amps of depolarizing current for a few seconds. A small crystal of dye eye will form on the cell to confirm that the cell has been labeled.
Briefly open the shutter to illuminate the field with fluorescent light. If the body of the cell shows signs of labeling, apply current for several more seconds. Then turn off the current and quickly pull the embryo away from the micro pipette using the stage controls.
If no D eye labeling was evident, the micro pipette may have been blocked and needs replacement. If the tip of the pipette snags other tissues on root to the cell and stains that tissue, try withdrawing the micro pipet slightly and reapproach the cell before resorting to replacing the micro pipette as needed. Continue labeling other cells in the same embryo or in other embryos on the same slide.
Move the tip to the edge of the field of view and repeat the procedure. Remove most of the solution in the injection chamber. Then fix the embryos by adding 7.4%formaldehyde PBS for 10 minutes, followed by four washes with PBS.
The preparation can now be photo converted or mounted for confocal microscopy using the outlined protocol. A dai filled inter neuron was flawlessly photo converted under DIC optics. The spatial context of the labeled cell within the non labeled surrounding tissue was well resolved.
For example, the positions of the cell body within the cortex and of the fiber projection within the neuro pill could both be seen in this preparation. The dye drop was a little too big. Several neighboring cells became labeled simultaneously, which makes it difficult to relate individual projections to distinct cell bodies.
Also notice that under the fluorescent microscope, the background resolution is much lower without photo conversion. Multiple cells in neighboring segments can also be injected in this preparation, an antibody against fast two stains of fales in the neuro pill. Despite the clear cell bodies, the preparation shows a possible side effect of prolonged photo conversion periods.
The more intensely labeled cells tend to get overs stained and start to swell as compared to labeling a single cell with confocal optics. Dye eye labeling in embryos that carry GFP reporter constructs can provide spatial context and identity of the D filled cell within specific populations of GFP positive neuronal or glial cell types. After watching this video, you should have a good understanding of how to prepare and perform labelings of single neurons in the embryonic CNS after oph.
But while attempting this procedure, please keep in mind that every single step is demanding, so expect a couple of repetitions until the whole procedure runs smoothly.
