The novel transgenic, IRFP Reporter Drosophila Line expresses red fluorescent protein or RFP in specific cells in the I imaginal disc. This transgenic line can aid in the identification, dissection, and labeling of specific cell types in the developing eye. To dissect the eye imaginal disc wandering third in star larvae are removed from the side of a vial using a spatula.
The larvae are then placed into a drop of PBS on a sill guard dish. The mouth parts with the eye discs attached are removed from the rest of the lava using tweezers, the eye discs are then moved to a slide where they're dissected away from the mouth parts and brain. Hi, I'm Amna Cole with the Joseph Bateman lab in the Wolfson Center for age related diseases, Kings College London.
Today we're going to show you a procedure for how to dissect imaginal eye discs from Drosophila. We use this procedure in our laboratory to study the genes involved in neurogenesis. So let's get started.
To begin dissecting eye imaginal discs, place a drop of PBS onto a syl guard coated Petri dish Under a dissecting microscope, the Syl Guard coating will help protect the tweezers from becoming damaged during the dissection. Next, using a spatula, take a wandering third in star lava and place it in the drop of PBS holding tweezers In each hand, grasp the lava two thirds of the way toward the posterior with one set of tweezers. Now use the other set of tweezers to hold the dark colored mouth parts at the anterior end of the lava.
Gently pull the lava apart by moving the tweezers away from each other. The mouth parts should come away from the rest of the body with the eye brain complex attached. Next place a drop of PBS on a slide and transfer the discs to the drop of PBS by holding the mouth parts.
Now switch to the fluorescence mode on the dissecting microscope since only the eye imaginal discs express RFP, they can be easily identified as red fluorescent tissue overlaying the spherical optic lobes to increase the contrast of the discs. Direct to fiber optic cold light source at an oblique angle to the slide. The eye imaginal discs are attached to the optic lobes via the optic stalk at the posterior and to the mouth parts via stalk coming from the an antenna disc at the anterior.
To dissect the eye imaginal discs, hold the parts using one pair of tweezers and use the other pair to separate the eye imaginal discs from the larval brain. No.Next, separate the eye imaginal discs from the mouth parts. Once the mouth parts have been separated from the eye imaginal discs, remove them from the slide.
The eye imaginal discs should now lie flat on its slide and should be clear of any extraneous tissue including brain tissue. Continue dissecting the remaining I imaginal discs. Once all of the eye imaginal discs have been dissected, gently tilt the slide and use a small piece of paper towel to dry off the excess PBS.
Now add a drop of vector shield mounting medium and gently place a cover slip on top. The discs are now ready to be imaged by either epi fluorescence or confocal microscopy. If the imaging will take place at a later time, store the slide at four degrees Celsius.
The RFP will remain visible for at least one year when imaging Z Series images should be acquired on a confocal or Zeiss aome microscope at one micrometer spacing using a 20 x or 40 x lens projection images can then be generated using the microscope software. Imaginal discs from flies expressing IRFP were dissected and stained with Phin Imaging was then performed on a zes aome microscope at one micrometer spacing using a 20 x lens. The cells of the eye disc, which express RFP are shown in red.
The morphogenetic furrow is visualized by foid and staining and is shown green. The dorsal ventral poles of the eye disc are curved and tend to fold. These sections can be emitted from the final projection image.
Although the disc is a monolayer epithelium, the variable positions of the nuclei require Z Series projection to get an overall picture of the disc. Since the IRFP reporter is nuclear localized, the pattern seen here represents nuclear expression of RFP. We've just shown you how to do the procedure to dissect vaginal eye discs from drosophila.
When using this procedure, it's best that you use a fine pair of tweezers and it's probably a good area to lay off the coffee as well. Thank you for watching and best of luck with all your experiments.
