השירות של שלב ספציפי אמצע לסוף

The overall goal of this procedure is to isolate specific stages of mint to late oph follicles for a variety of experiments, including molecular analyses, live imaging, and in vitro development assays. This is accomplished by first dissecting out the ovaries and then separating the ovaries into ols. Then once specific stage follicles have been isolated, they can be utilized for molecular analyses, live imaging, or in vitro development.

Ultimately, results can show what aspects of late drosophila oogenesis are regulated by specific factors through pharmacologic and genetic manipulations in combination with these assays. This method can help answer key questions in the fields of development and cell biology, such as acton, cytoskeletal dynamics, morphological processes, tubular genesis, and extracellular matrix secretion and patterning. For this protocol, collect well-nourished young adult flies of both sexes two days before isolating the follicles.

If the stage follicles are going to be used for in vitro development, assays have freshly prepared Ivan Media ready at room temperature. Cold media will alter cytoskeletal dynamics and development. If the stage follicles are going to be used for mRNA or protein isolation grace's insect media is sufficient.

Now begin by anesthetizing flies in their vial with an injection of carbon dioxide, and then transferring three to five females onto a carbon dioxide emitting fly pad. Be aware that the flies cannot remain under CO2 any longer than 10 minutes without harm. Now place a nine spot plate on top of a dark surface like black plexiglass.

Add media to one well and focus the dissecting scope on the bottom of the well. Using number five Dumont forceps in your dominant hand, pick up a single female by the wings, legs, or transition between the thorax and abdomen. Submerge the female in the media and keep her in focus.

Now with another pair of forceps, grab the female at the anterior end of the abdomen so that the posterior end is positioned towards the dominant hand. Make sure to keep the fly submerged with the dominant hand. Use the forceps to grab the cuticle of the second most posterior pigmented segment.

Then rip it away from the rest of the abdomen. Using the forceps in the dominant hand, gently squeeze the anterior end of the abdomen until the ovaries come out. If necessary, place the forceps in between the two ovaries and pull them out of the carcass.

Next, move the ovaries to a new well with fresh media or remove the carcass from the well. Repeat this process until three to five pairs of ovaries have been isolated. Take note that each ovary is composed of about 16 ovial chains of sequentially maturing follicles.

Also note that each of the ovial is contained within a muscle sheath. Once the ovaries are isolated, use pin vices and the associated needles to gently pull apart the ovary. By running the needles between the ovial individual, ovial should now be visible and sometimes individual follicles get released.

Once the ovial are separated, the different stages of follicle developments can be easily identified. Refer to figure one in the accompanying text for a printable and detailed description of the distinguishing features and anatomy of each stage. To isolate follicles contained within their muscle sheath, use a needle to cut across the ovial at the anterior of the proceeding follicle and at the posterior of the follicle immediately following the follicle of interest.

As individual stages are separated, move the follicles of interest using a glass pipette to a new well with fresh media. Once enough follicles have been isolated, proceed with subsequent experiments To ensure the greatest possible success when using the in vitro development assay. Two things are critical.

First, that the i VM media is at room temperature, and second, that the dissected ovaries and follicles are rapidly removed from the debris filled wells during the dissection. Be extremely careful to separate the stage 10 B follicles from the stage 10 A follicles because the 10 a follicles will not mature in the IVE culture media. Thus collecting stage 10 A follicles will skew the results in both stage 10 A and stage 10 B follicles.

Half of the length is composed of nerve cells and the other half is the oocyte, but in stage 10 B, the length of the follicle is equal to that of a stage 14 follicle. Make sure that the follicles are rapidly moved away from debris as these follicles will continue to mature. It is important that stage 10 Bs are collected for 30 to 60 minutes and then placed into the maturation media of choice, verify that all of the follicles being transferred are stage 10 B and have not matured to stage 11.

Using a glass pipette move about 30 stage 10 B follicles into a well of a 24 well tissue culture plate. Using a polled glass pipette remove as media from the well as possible, quickly replaced the media with a milliliter of maturation media, ive mixed with the selected pharmacological reagents. Continued loading as many wells with stage 10 B follicles as the experiment requires now allow the follicles to develop for at least 10 hours at room temperature, which is typically overnight.

Finally, score the number of follicles at each stage of development. Typically, follicles have completed nurse cell dumping. If they have matured to stage 12, 13, or 14.

Alternately, they have failed to complete dumping if their development has stalled at stage 10 or 11. The development of stage 10 B follicles in culture is robust. In a successful experiment in 80 to 100%of the wild type follicles in control media will complete nurse cell dumping.

However, several factors can mar the results improperly. Prepared media inability to distinguish stage 10 A from stage 10 B or prolonged exposure to debris will prevent wild type controls from developing. In a pharmacogenetic experiment, one genetic background represented by the blue bar fail to enhance or suppress follicle sensitivity to the IC 50 of aspirin.

While another background represented by the red bar showed a strong enhancement of follicle sensitivity to the effects of aspirin in pharmacological experiments, drug concentrations can drift over time using a wild type control. Aspirin was found to become too concentrated because the IC 50 treatment blocked greater than 50%of the control follicles from developing stage Isolation can also be used for live imaging of developmental processes. For example, in a stage 10 B follicle expressing GFP fu, see the actin binding domain of utrophin actin bundle formation and condensation was visualized.

Finally, individually, stage follicles can be pulled together for molecular analyses, including western blotting and microarray analyses. After watching this video, you should have a good understanding of how to isolate mid to late stage eslo follicles for use in in vitro development assays, live imaging, and for molecular analyses.