This procedure begins with culturing fat crawling drosophila third in star larvae in a fly bottle. Using two pairs of forceps, the salivary glands are dissected away from the larvae. The salivary glands are then successfully fixed in two different fixatives in a two well depression slide.
Finally, the glands are squashed in a lacto acetic acid solution between a cover slip and a microscope slide. Hi, I'm Dr.Kristen Johansen in the Department of Biochemistry, biophysics and molecular biology at Iowa State University. Hi, I am TA graduate student in the Johansen's lab.
Today we're gonna show you a procedure for making drosophila polytan chromosome squash preparations suitable for antibody labeling. We use this procedure in our laboratory to study epigenetic histo modifications and the relationship to chromatin structure and gene expression. So let's get started.
In order to obtain optimal polytan chromosomes for high quality squash preparations, uncrowded culturing conditions are essential. Place around 20 egg laying female flies in a standard four inch fly bottle and change to a new bottle each day. Culturing at 21 degrees Celsius is fine for most purposes, but incubation at 18 degrees Celsius will yield fat or chromosomes that may be more suitable for some experiments, such as when banned interband regions need to be visualized at high resolution.
Select the fattest individuals from the first crop of climbing third in star larvae. While they are still wandering, but just prior to pation, the larvae are easily visible on the side of the bottle above the food. Use forceps to place larvae on a tissue culture dish.
For each round of squashing, rinse two to three of the larvae together with water before transferring them to a clean dish of PBS for dissection. The PBS should cover the larvae, but time is not of the essence. Since the larvae can survive under these conditions for hours, place the dish of larvae in PBS on the stage of a dissecting microscope with one pair of forceps.
Grasp the tip of the mouth hooks while holding the body. About two thirds of the way down with a second pair, pull on the mouth hooks with the forceps to expose the salivary glands. Separate the salivary glands from the brain and eye and tenal discs and dissect away the fat body and any other associated tissues.
Add 200 microliters of fixative one to the first well of a two well depression slide and 200 to 300 microliters of fixative. Two to well two. Transfer one pair of salivary glands at a time to fix it of one in well one and incubate for the amount of time required for the target epitope usually about one to two minutes using forceps.
Transfer the salivary glands to fix it of two in, well two and incubate for two minutes Using forceps, transfer the glands to a clean sigma coated cover slip with 10 to 30 microliters of freshly prepared lacto acetic acid solution. Gently lower a polylysine coated microscope. Slide onto the cover slip.
Carefully lift the slide using the bonding of the liquid to the glass to pick up the cover slip without applying vertical pressure. To the preparation to spread the chromosomes immediately and carefully grasp the edge of the cover slip with forceps and gently move it slightly back and forth. Again, minimizing vertical pressure on the tissue, the wiggling of the cover slip is important for adequately separating the individual cells of the salivary gland.
Gently tapping the cover slip obliquely with the eraser side of a pencil may also assist in spreading clouding of the solution is often a good indication of cell separation. Promptly examine the tissue under a phase contrast microscope to check that the chromosomes are well spread as indicated by clearly separated and extended chromosome arms. If the chromosomes do not appear, well spread the first time.
It is possible to repeat the gentle tapping of the cover slip until sufficient spreading is obtained. Set the slide cover slip side down on a stack of clean Kim wipes. Place a clean wipe on top of the preparation.
Place a thumb over the position of the cover slip and firmly press straight down. Avoid horizontal movement, which would shear the chromosomes. Reexamine the slide under the microscope to determine suitability for the current experiment.
Repeat the dissection and chromosome squash preparation to obtain a sufficient number of slides. Place a 175 gram weight on the cover slip of the slides using long forceps. Dip a prepared slide into a small doer of liquid nitrogen and hold it there until the boiling stops.
Remove the slide and immediately use the edge of a clean razor blade under the corner of the cover slip to flip it off. Collect the slide into a coplan jar filled with cold PBS for immediate use, or filled with 95%ethanol for longer storage. Once the frost has sublimed away, examine the cover slip to confirm that the tissue remained with the slide.
The first critical step for obtaining high quality INE squash preparation is to grow fat larvae with big salivary gland nuclei. The second is good technique with the spreading procedure, which may take some practice. One tip for improving spreading success is to find the minimal volume of lacto acetic acid solution during the squashing step.
This will promote sufficient spreading of chromosomes without generating excessive streaming forces that can wash chromosome arms away. It is also worth noting that any delay in moving the cover slip back and forth will reduce spreading of the chromosomal arms as they become more rigid when exposed to the lacto acetic acid solution. If everything goes well on each slide, there should be numerous well spread polytan chromosomes.
Here we show an example of such a preparation double labeled with a marker for interband regions in red and with a dye which stains the banded regions in blue. If insufficient spreading is obtained, the chromosomes will look like little balls as shown here. On the other hand, if too much spreading has occurred, the chromosomes will be too thin and extended or in some cases fragmented into small pieces as shown here.
So we've just shown you how to make drosophila poly team chromosome squash preparations When doing this procedure. It is important to remember to start with fat la. So that's it.
Thanks for watching and good luck with your experiments.
