We dissect the larvae by grasping the posterior and anterior ends, then pulling the forceps in opposite directions. Excess tissue is trimmed away using ultra fine scissors. This will reveal the two hemispheres of the larval brain eye imaginal discs.
The ventral nerve cord and mouth hooks. The larval eye brain complex is transferred into a culture chamber. We separate the brain lobe from the optic stalk to visualize the migration of glial cells within a wild type eye imaginal disc.
To observe stalled glial cells in the optic stalk of a mutant, we leave the brain lobe attached to the optic stalk. Hi am Patrick Cafferty from Vanessa Als Laboratory in the Department of Zoology at the University of British Columbia. Hi, I'm Rin.
She also from Today, we'll show you a procedure for visualizing migrating glial cells in the developing drosophila eye. We use this procedure in our labate to study glio cell development. So let's get started.
One week prior to the start of the experiment, flies are mated to generate larvae that expressed GFP under the control of a glial specific promoter. This experiment involves visualization of GFP tagged with a nuclear localization sequence expressed in glial cells. Using the reversed polarity promoter prepare 18 millimeter round cover slips at least one day in advance by soaking them for 10 minutes in 1%poly L lysine solution, and then air drying them overnight on the day before the experiment.
Clean a chain lied magnetic culture chamber by soaking its components overnight in 70%ethanol on the day of the experiment. Use aseptic techniques to prepare culture medium. Add fetal bovine serum penicillin, streptomycin solution, and insulin to 10 milliliters of Schneider's insect medium.
Allow the clean chain lied magnetic culture chamber to air dry in a cell culture hood before washing off residual ethanol by rinsing with prepared culture medium. Once these preparations have been made, we can begin the dissection of the drosophila eye brain complex. To begin this procedure, select a third instar wandering larvae from the side of a fly vial and place it in a drop of chilled culture medium on a Petri dish on ice for several minutes chilling.
The larvae is recommended to slow peristaltic body contractions for ease of dissection. Next place the chilled larvae in a drop of culture medium on a sard coated dish under a dissection microscope under the dissection microscope, use a pair of dumont fine forceps to firmly grasp the larvae. Approximately one third of the way from the posterior end, wait until the larvae extrudes its mouth hooks from the anterior end and when the mouth hooks are fully extended, grasp them with a second pair of forceps.
To dissect the larvae, slowly pull the two pairs of forceps in opposite directions. The eye brain complex, along with salivary glands, fat bodies and imaginal tissue will pull away from the larval body. Don't pull the mouth hooks too quickly or they will tear off of the iBrain complex.
Trim away salivary glands, fat bodies, and imaginal discs Using ultra fine clipper scissors, the two hemispheres of the brain and eye discs will be attached to the ventral nerve cord and mouth hooks. Next place an 18 millimeter cover slip onto a glass slide, leaving one edge of the cover slip hanging off the edge of the slide. In order to pick up the cover slip later, add a drop of culture medium onto the cover slip.
Grasp the mouth hooks with forceps and transfer the iBrain complex into the culture medium on the cover slip. Once the eye brain complex has been transferred to the cover slip, use fine dissection. Scissors to trim off the mouth hooks from the eye brain complex.
Removal of the mouth hooks is important for live imaging as the mouth hooks will continue to contract in the culture medium causing the tissue to move during microscopy. To visualize GL within the optic stalk, leave the brain optic stalk and eye imaginal disc intact. To visualize glial migration within the eye imaginal disc, carefully cut the optic stock, the thin tissue that connects the brain and the eye disc and push away or discard the brain.
Finally, pick up the cover slip using forceps and draw a circle underneath around the tissue of interest with a permanent marker. This circle will aid in locating the tissue for microscopy later. Now the eye imaginal disc is ready for mounting in a magnetic culture chamber.
To mount the eye imaginal disc in a magnetic culture chamber, use forceps to grasp the hanging edge of the cover slip and transfer the cover slip to the bottom plate of the chain line magnetic chamber without disturbing the tissue in culture. Next, place the silicone o-ring onto the main body of the chain line chamber. Install the main body on top of the bottom plate slowly.
Add culture medium to the chamber. Don't add the culture medium too quickly or the tissue will be disturbed to allow for gas exchange under the cover. Don't fill the chamber completely gently place the invisible cover on top of the chain line chamber.
To visualize migrating glial cells, place the culture chamber on the stage of a confocal or fluorescence microscope. Locate the sample using the circle on the cover slip as a reference on low magnification. Focus on the sample using a 40 times lens.
Allow the tissue to settle and capture images of the eye disc or optic stalk every 10 to 15 minutes over three to four hours if performed correctly. This protocol will result in a series of images of GFP tagged glial cells migrating from the optic stalk into the eye imaginal disc. While live imaging for a 60 minute period was sufficient to observe changes in the positions of glial nuclei with a wild type eye imaginal disc glial nuclei in a mutant for a gene necessary for glial cell migration completely failed to exit the optic stalk.
We have cultured eye brain complexes for periods as long as 240 minutes before observing deterioration of the cultured tissue. Following the 240 minute time point, we begin to observe GFP positive cells in the culture medium surrounding cultured eye brain complexes. In addition, GFP will accumulate in diffuse spots throughout the tissues suggesting a breakdown in tissue integrity.
We've just shown you how to culture. I imaginal discs in order to study glial cell migration. Thanks for watching and good luck with your experiments.
