נתיחה והתקנה<em> דרוזופילה</em> גלמי דיסקי העין

Welcome to a pupil eye disc dissection and mounting of drosophila melanogaster. An appropriately HD pupa should be selected. The dissection begins by piercing the pupil case and removing the pupa from the case.

A cut is made diagonally mid thorax in the pupa. After cutting the pupa, the brain and iis will be isolated and processed prior to mounting the iis. For microscopy to begin the dissection process and appropriately age pupa is selected.

Place the pupa in a large drop of PBS on the dissecting dish. The best practice is to orient the pupa with the mouth hooks facing downward. These are the mouth hooks facing the mouth hooks downward.

Grasp the pupa gently with one pair of forceps. Being careful not to squeeze the developing pupa inside the pupil case too hard with a sharp pair of forceps. Pierce the pupil case and let the four steps spread to tear off the end.

Reach inside and grab the developing pupa out of the hole that was just created. Next, take a pair of scissors and cut the pupa about mid thorax level. Make the cut on a diagonal.

Then it is best to remove the debris or transfer the PU ahead to a new drop of PBS. Cleaning can be done using the pipette blower tube and additional PBS should be added as needed, making sure to avoid dehydration. Next securely grip the top of the pupil head cuticle with a pair of forceps.

Be sure to avoid grabbing any tissue within the cuticle using the tip of the blower tube. Gently push on the pupil case to extrude the brain and attach eye disc out of the pupil case. To aid in this process, use PBS in the blower tube to gently blow the debris as seen here out of the head case to allow the clear separation and identification of the brain and I discs continue in this process of pushing and blowing as necessary until the brain and I discs are separated from the surrounding tissue and easily identified the I disc can now be seen here and this pupa pigmentation has already begun and the I diss have a slight color.

The eye diss are outlined in red and the brain is highlighted in between. At a higher magnification, each I discs can be easily identified. Again, the I discs have been outlined in red with the brain in between.

The brain and I.Discs should continue to be cleaned and separated from the remaining tissue by using forceps and the blower tube optimally, the eye disc and brain should still remain attached to the head cuticle so that it may act as a handle for the forceps for future manipulation. Although through the blowing and pushing process, it is common to dislodge the brain and eye disc from the head cuticle. After careful isolation of the I dis and brain gently place the tissue into the fixed solution.

Initially let the I disc float on top of the fix so that they don’t collapse on themselves and get fixed In a closed orientation, the best practice is to dissect the I dis and let them float on the fix. They remain floating while the next set of I discs are dissected, and then they are submerged into the fix following fixation of the I disc. They’re processed as needed, such as immuno staining and then mounted on a slide for visualization.

Here are fixed pupil I discs with the brain still attached in a large drop of PBS. Mounting and glycerol is very difficult to do, although we have successfully mounted iis and 50%glycerol and PBS. If you mount in PBS, be sure to avoid dehydration.

Notice that the head cuticle has been removed. Outlined in red is each I disc. The brain is highlighted in between.

Take sharp forceps and sever the connection between the eye disc and the brain. This is accomplished by holding the brain and placing the sharp forceps at the connection here. This can be very difficult.

It is important to use sharp forceps because they make this process much easier. It is also possible to make this separation using a fine dissecting needle as well as can be seen. Sometimes it is easier to pull the brain off while stabilizing the eye disc at the connection.

There is quite a bit of extraneous tissue left on this eye disc that should not affect the imaging of the eye disc as long as the I disc is mounted on the microscope slide with this side facing up. This allows for the apical surface of the I disc to be flat on the microscope slide. We have found that further removal of this tissue usually leads to tissue damage of the I disc.

So does preferable to leave this tissue on and avoid it in the imaging stage. Another technique to remove the brain involves pinning the eye disc at the connection with either a dissecting needle or a sharp pair of forceps as seen here and then pulling the brain away. This detachment was much cleaner with less tissue remaining on the I disc.

When you have finished preparing all your I discs, line them up in a row with the optic track oriented upwards. Remove the PBS and replace it with VTA shield or other mounting media and cover them with a cover slip. The I discs are now ready to be imaged through the techniques described here along with the detailed immuno staining protocol.

In the manuscript I disc can be visualized with confocal microscopy for morphological characteristics as seen here with the phosphotyrosine staining, revealing the apical morphology of the omel cells in the I disc. Other antibodies and genetic techniques can be employed to identify cell pipes and expression patterns as seen here for the expression of the cut transcription factor in the cone cells of the I disc. This video represents a remastering of training videos that were produced to instruct undergraduate students in this technique.

Many of our students were successfully trained in this technique using this video and we hope that it will assist you as well. We wish you the best of luck in your experiments and hope that this technique will be a benefit to you.