In this protocol, cytes expressing GFP tagged proteins are dissected from female drosophila and imaged to study the movement of the protein in living cells. First, prepare a viewing chamber by affixing two cover slips to a slide using silicone grease to form a channel. The sides of the channel are lined with halo carbon oil.
Next, dissect the ovaries from a female into a drop of halo carbon oil on a cover slip. Then isolate the individual cytes and invert the cover slip over the channel in the prepared slide to create a viewing chamber. After locating the cytes with brightfield or DIC optics, capture digital images at set time intervals using confocal microscopy and associated software.
Ultimately, a time-lapse sequence of the images generated can be used to monitor the movement of proteins or particles in living cells over time. This method can help investigate key areas in cell and developmental biology, such as protein and mRNA trafficking, and the establishment of cell polarity. First anesthetize the healthy flies that are less than one week old and select 10 to 15 large females with rounded cream colored abdomens.
Transfer the selected flies and a few males to a vial containing new lightly yeasted food. Then incubate the flies for two days at 25 degrees Celsius to fatten. Fattening the flies ensures that a variety of stages, especially stage eight through 12, are available for imaging un fattened flies or poorly fattened flies usually only contain very early stages and mature cytes Using silicone grease.
Affix two cover slips approximately one centimeter apart to a standard glass slide. Only use enough grease to ensure a good seal between the cover slip and slide. Press down firmly on each cover slip to spread the grease thinly and evenly.
Then add some hallow carbon oil 27 to the juncture between the cover slips and slide to prevent injuring isolated cytes in subsequent steps. Now place two to three drops of carbon oil 27 onto the surface of a 22 squared millimeter cover slip. Next mouth pipette an individual female from the food vial and gently deposited into the halo Caron oil.
Using sharp dissecting forceps, pin the fly against the cover slip and pull off the tip of the abdomen. Remove the ovaries by dragging them along the surface of the cover slip under the oil to promote adhesion of the cytes to the cover slip. Now gently tease apart the ovaries looking for cytes that are at least stage 10 B of Oogenesis.
Identify oocytes at this stage by looking for egg chambers in which the oocyte occupies at least 50 to 60%of the total volume of the egg chamber. Using forceps. Remove individual oocytes from the ovial sheath using one to three females.
Continue to isolate five to eight undamaged oocytes on the cover slip. Then remove any unusable tissue. Carefully invert the cover slip containing the oocytes onto the pre-prepared slide so that the cytes are positioned between the two Spacers.
And remember, do not press down on the cover slip as it can damage the cytes if Necessary, add a small amount of halo carbon oil to the edges of the sandwich to ensure the space is filled. Notice that at this point in oogenesis, the follicle cells overlying the oocyte have become coer and surround the oocyte on all sides, except for a small region where connections to the nurse cells remain. Bring an oocyte into focus using DIC or phase contrast optics.
Examine the oocyte for any signs of damage, such as leaking of cytoplasm or bulging of follicle cells. Image only the oocytes that appear healthy and undamaged. To monitor streaming directly without GFP labeling.
Set capture images in the ZI range of the scope with higher gain settings than those used for GFP labeled proteins. Using air objectives to greatly minimize drift and movement of the sample. Obtain optical sections just below the surface of the oocyte.
Once a region of interest is in focus, turn off the brightfield optics and switch to confocal imaging using the fast scan preview function of the software, adjust focus and gain as necessary. Also, set the parameters for excitation, wavelength, and an emission wavelength. For the Z axis capturing, select a constant Z axis with a lag of 10 seconds between frames.
After capturing a typical time lapse sequence of between 20 and 50 frames immediately review and assess the video quality. This single time lapse image shows a stage 11 oocyte. This oocyte expressing the GFP tagged protein R TNL one can produce a strong signal.
R TNL one appears to co localize with the long microtubules present during op plasmic streaming. Typically, op plasmic streaming in a wild type egg chamber is evident as noticeable movement of autofluorescent ylk vesicles as shown in a five frame maximum projection of a stage 10 B egg chamber. After watching this video, you should have a good understanding of how to isolate healthy female cytes expressing GFP tagged proteins, how to prepare samples for live imaging, and how to take time lapse videos of fluorescent proteins and particles.
