Upright Imaging of Drosophila Egg Chambers

The overall goal of this procedure is to enable high resolution imaging of the poles of drosophila egg chambers. This is accomplished by first dissecting ovial and staining them with fluorescent antibodies. The second step is to identify and separate egg chambers of interest.

Next selected samples are oriented and aligned, then embedded in glycerin jelly blocks. The final step is to cut and rotate blocks to mount egg chambers so the long axis is vertical. Ultimately, confocal microscopy is used to reveal cellular and subcellular organization at the poles of the egg chamber.

Epithelium, such as border cell arrangements, Existing methods to view specimens perpendicular to the imaging plane via optical sectioning and algorithmic 3D reconstruction can cause photobleaching and light scattering and provide poorer resolution, which yields less accurate images. The main advantage of this technique is that it allows us to mount specimens at a new angle and view them directly. We use this technique to image the poles of developing egg chambers, which provided insight into the organization of follicle cells.

Visual demonstration of this method is critical as the preparation and mounting steps are difficult to learn because they require micro manipulation of stain specimens and reorientation of the samples from the conventional viewpoints. Demonstrating the procedure will be Lathea manning a graduate student in my laboratory. To begin the experiment, obtain a glass depression slide and pipette several drops of dissection media into each cavity.

Anesthetize the flies with carbon dioxide and place them under a dissection microscope. Next, orient one female fly with the wings down and the head towards the left. Hold the forceps in each hand at a 30 degree angle to the tabletop with the non-dominant hand.

Pick up the fly using forceps. Grasp the fly at the anterior of the abdomen near the thorax and submerge it into the dissection media. In the depression slide with the other pair of forceps.

Grasp the exoskeleton at the ventral posterior of the abdomen and pierce through. Grab the ovaries at the posterior end, then pull them out of the abdomen and place them into the fresh media on the other side of the depression slide. Then hold an ovary at the posterior end near the largest egg chambers, and with the other hand, pinch one of the most anterior egg chambers or jamer slowly and steadily.

Pull an ovial chain out of the ovary sheath and continue to dissect out the ovial individually until all desired egg chambers are removed. Once dissection is complete, use a plastic transfer pipette to transfer the ovial to a 0.6 milliliter tube and let the ovial settle to the bottom of the tube. Using a pipette carefully remove as much of the dissection media as possible from the ovial.

Be careful not to remove any ovial or egg chambers in a new tube. Add 50 microliters of 16%para formaldehyde to 150 microliters of 0.1 molar potassium phosphate buffer, and add this to the egg chambers. Incubate them while rocking for 10 minutes After incubation, remove the fixative.

Rinse twice with NP 40 buffer, then washed over aerials two times with 0.5 milliliters of NP 40 buffer for 15 minutes each at room temperature. After fixation and washing, add primary antibodies and incubate the ovial overnight at four degrees Celsius or three hours at room temperature Following incubation. Wash the samples four times for 20 minutes each with 0.5 milliliters of NP 40 buffer at room temperature.

Then add secondary antibodies at a one to 200 dilution and incubate the ovial for three hours or overnight at four degrees Celsius. Next, add DPI at a one to 1000 dilution and incubate the ovial for 10 minutes. Then wash the samples four times with 0.5 milliliters of NP 40 wash buffer for 20 minutes per wash at room temperature.

Once the immunofluorescence or IF protocol is complete, add 200 microliters of 50%glycerol in PBS to the egg chambers and let them sit at room temperature for two hours protected from light. Remove the solution, then replace it with 70%glycerol in PBS and cover the tube with foil. Store the samples at four degrees Celsius until they are ready for mounting.

To prepare the glycerin jelly slide aliquot glycerin jelly with a spatula to fill a 15 milliliter Pyrex glass culture tube about halfway melt the glycerin jelly in a water bath at 55 degrees Celsius for 30 minutes. Meanwhile, place a 300 microliter drop of glycerol from the stock solution onto two microscope slides. Using a tissue, spread the glycerol in a thin layer across each of the slides.

To provide a base that enables easy removal of glycerin jelly. Check that the glycerin jelly is thoroughly melted. Next, cut the tip off of a filtered 1000 microliter pipette tip and transfer 1000 to 1, 500 microliters of warm glycerin jelly slowly to each microscope slide taking care to prevent bubble formation.

Let the glycerin jelly slides sit for five minutes at room temperature to set lace each glycerin jelly. Slide in a 60 millimeter by 15 millimeter Petri dish. Cover the dish with plastic wrap and store the slides at four degrees Celsius overnight.

Pipette several three microliter drops of egg chambers from the previously prepared samples in 70%glycerol across one glycerin jelly slide. Continue pipetting three microliter drops until all of the egg chambers are on the slide, making sure each drop contains at least 10 egg chambers. Next, heat to culture tube of glycerin jelly in a 55 degree Celsius water bath, placed a glycerin jelly slide with egg chambers under a dissection microscope and adjust a magnification so that only one drop of egg chambers is in the field of view.

Using a 30 gauge needle as a spoon, pick up the desired stage egg chamber when necessary. Separate desired egg chambers from an ovial chain using the needle as a knife to avoid debris that may interfere with imaging. Transfer the egg chamber to the second glycerin jelly slide.

Repeat this step until there are at least seven egg chambers lined up in a column with the anterior sides facing left. Scrape away debris or unwanted egg chambers with the needle form new columns of seven until all desired egg chambers are transferred to the second glycerin jelly slide twist a small piece of Kim wipe, then absorb the excess PBS glycerol from the egg chambers by lightly touching each one. Take care to not remove any egg chambers.

Cut off the tip of a filter 200 microliter pipette tip and transfer 150 microliters of glycerin jelly to cover all of the columns of the egg chambers. Verify under the microscope that all egg chamber columns are covered. Let the mounted egg chambers sit for five minutes at room temperature until the top layer of glycerin jelly has completely solidified.

Then place a slide of mounted egg chambers in a 60 millimeter by 15 millimeter Petri dish and cover it with plastic wrap. Store the dish at four degrees Celsius overnight to allow the glycerin jelly layers to solidify together in the dark. If photobleaching could occur, place a slide of the mounted egg chambers under a dissection microscope.

Adjust a magnification so that only one column of egg chambers is in the field of view. Using a 45 degree angle miniature scalpel. Cut a straight line along the right side of the first column of egg chambers close to the posterior side of the egg chambers.

Next, cut above the first egg chamber at the top and below the last egg chamber in the column so that the egg chambers are now cut on three sides. Use a micro knife to slice along the left side of the column as close as possible to the anterior side of the egg chambers. Then pipette 100 microliters of cold one XPBT over the cut column with a round and its spatula.

Remove the excess glycerin jelly around the three sides of the cut column of egg chambers and discard the excess. Insert the spatula in the cut made at the anterior side of the egg chambers and separate the column from the primary glycerin jelly block to mount on a slide and proceed with imaging. The upright imaging method enables a direct visualization of the organization of cells in the anterior follicular epithelium at stage eight.

A general marker for follicle sulfate, the eyes absent or IA protein, as well as the nuclear DNA marker DPI show even expression across this field of cells and demonstrate that all cells can be seen with similar staining intensities. The conventional view of an egg chamber contrasts with the on view created by a three-dimensional reconstruction of the egg chamber. The inset shows the turned axi.

The x axis is down. Z axis is now towards the left, and the Y axis is towards the viewer. Individual cells are blurred due to low resolution in the Z axis.

Thus the new approach represents a significant imaging improvement. The upright imaging method effectively visualizes the anterior epithelium in stage nine egg chambers. At this developmental stage, the border cells coalesce together around the polar cells.

These compacted clusters are observed with the endon imaging. After watching this video, you should have a good understanding of how to mount samples to enable new viewing perspectives. We have used this technique to image the poles of developing egg chambers at selected stages.

This general technique could be used for other stages of drosophila, oogenesis, or for other types of small tissues.