Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

The overall goal of this procedure is to use DNA in situ hybridization to map hetero chromatic sequences to mitotic chromosomes. This is accomplished by first dissecting brains of third in star drosophila larvae, or another tissue of interest. The second step is to fix the tissue on a cover slip.

Next, the tissue is squashed between the cover slip and a slide. To prepare the chromosomes for hybridization, the final step is to hybridize short synthesized oligo probes to the squashed tissue. Ultimately, fluorescence microscopy is used to show the localization of sequences complimentary to the oligo probes on the chromosomes.

The main advantage of this method over existing methods is its simplicity. Most other chromosome and C two hybridization methods involve washes with large amounts of form amide, which can be expensive and messy. Our method requires no form IDE washes, making it a cheaper, cleaner, and faster technique.

Although this method provides a simple way to visibly map sequences on the somatic chromosomes of drosophila brains, it can also be used to map sequences on other types of chromosomes from different tissues in the different organisms. For each slide to be made, prepare four or five tissues, but not more for brain dissections. Begin with selecting wandering third instar larvae and collecting them in a drop of one XPBS.

Choose vials to collect from that are not overcrowded. Under the dissection scope, use ultra fine tweezers to grab hold of the mouth hooks and another pair to grab two thirds down the length of the body. Then gently pull on the mouth hooks to expose the brain, ventral ganglia, salivary glands, and parts of the digestive tract.

Next, tweeze the brain and ventral ganglia away from the other tissues and transfer them into a droplet of one XPBT on a plastic plate. For nasia testis dissections collect male three day old puy. Three day old puy have yellow bodies and red eyes.

The ones with smaller wing pads are male. Grip the pupa at the top of the abdomen near the thoracic region with one pair of tweezers and with a second pair of tweezers. Grab the very distal tip of the abdomen, then pull out the teardrop shaped testes.

They are surrounded by fat bodies that can be gently shaken away, detach any exterior body parts from the testes, which will prevent proper squashing and transfer the testes to a drop of one XPBT on a clean plate. Once all the tissues are collected, treat them with a hypotonic solution to improve the separation of sister chromatids. In the e chromatic regions, transfer the tissues to a drop of 0.5%sodium citrate for 10 minutes.

To get this effect, colchicine is not recommended because it can cause artifacts in chromosome morphology. Now, fix the tissues using ultra fine tweezers. Transfer them into a 20 microliter drop of freshly made 2.5%PFA in 45%acetic acid on a clean sigma coat treated cover slip space the tissues out evenly in the fixative and incubate them for four minutes at room temperature and then proceed with squashing the cover slips begin by lightly placing a polylysine coated slide face down onto the tissue and cover slip.

Allow the cover slip to stick onto the underside of the slide without pressing.Treating. Cover slips with sigma coat helps to ensure that the squash tissue remains on the slide following cover slip removal. However infrequently the tissue could remain attached to the cover slip.

During this step, we recommend performing replicate tissue squashes. Now flip the slide over and place it into a folded piece of thick paper. Then on a stable surface, using the thumb press very firmly straight down over the cover slip.

Do not press in a way that slides the cover or it will smear the tissue. Very firm pressing normally results in nicely resolved chromosomes within the squash tissue. However, on occasion, you may encounter variability in chromosome quality such as incomplete, squashed, or fragmented chromosomes.

So again, we recommend performing two or three replicate tissue squashes. Transfer the assembly to a block of ice for a few minutes, then submerge it into liquid nitrogen until the nitrogen stops boiling or longer After removing the slide immediately before the tissue thaws, use a fresh razor blade to flick up a corner of the cover slip. Thus removing it.

Be careful to avoid scratching the tissue. Then quickly place the slide into a coplin jar filled with 100%ethanol, and let's soak for at least five minutes. Remove the slide wick away excess ethanol without touching the tissue and let the slide air dry for an hour.

Slides can then be stored for weeks to months in a desiccation chamber. Begin with adding 100 nanograms of each probe to 20 microliters of 1.1 x hybridization buffer for a 22 microliter solution of one x hybridization buffer. Then pipette the mixture onto the fixed tissue, leaving the tissue untouched and gently apply a cover slip.

A small air bubble not contacting the tissue will not present problems, but any more bubbles, warrants reapplication of the cover slip. Set the slide on the flat side of a 95 degrees Celsius heat block and cover it with foil. Wait five minutes during which you can prepare the humidity chamber by adding wet paper towels to a tip box.

Then remove the slide. Carefully wrap the slide in stretched paraform and transfer it to a humidified chamber composed of a moist towel In a tip box, incubate the slide in the chamber for four hours to overnight at 30 degrees Celsius. This temperature works well for low GC content probes, but for probes with higher GC content, some troubleshooting with hybridization temperatures may be required.

After the incubation, carefully remove the para film and slowly lift the cover slip by one corner. Then wash the slide three times in 0.1 XSSC buffer for 15 minutes per wash. Cover the jar with foil during the washes to minimize light exposure.

For long biotinylated probes, add rod domine avadon and wash before proceeding. Firstly, dry the area around the tissue and cover the tissue in 100 microliters of blocking solution. Then gently apply a new cover slip with without trapping bubbles.

Again, wrap the slide with param. Then incubate the slide at 37 degrees Celsius for 30 minutes. Secondly, after the incubation, remove the cover slip and blot around the tissue with a wipe.

Then onto the tissue, apply 100 microliters of one to 1000 rumine avadon diluted in SBT. Follow up by covering, wrapping, and incubating the slide as done with the block. Lastly, carefully remove the cover slip and wash the slide.

Use three washes in four x SSCs T, followed by three washes in 0.1 XSSC. Each wash step should be at least five minutes long. Proceeding with the protocol, dry the slides by blotting away the excess buffer without touching the tissue.

Then place the slide tissue side up in a dark place for 10 to 15 minutes or until the moisture completely dissipates. Now, mount the slide using 11 microliters of vector shield with DPI and carefully lower on a non sigma coat cover slip. If the medium does not reach all the edges, apply a few more microliters of media from the edge and wipe away any excess.

Then seal the cover slip with nail polish and store the slide upright in a dark place while the nail polish dries, which usually takes at least half an hour. Then store the slide at minus 20 degrees Celsius for up to a week and image the tissue as soon as possible. A set of small commercially synthesized oligos with fluorescent conjugates and a longer biotinylated probe made by Nick Translation were all hybridized to chromosomes from several different tissue types.

Using the described methodology, the probe to the Drosophila Melanogaster 359 base pair satellite repeat recognized a large multi megabase pair block on the x and a much smaller region on chromosome three. A longer probe against the responder satellite detected repeat blocks consisting of relatively low repeat copy numbers of about 700. Thus, this method allows visualization of very small satellite blocks.

When the hypotonic solution treatment step was doubled in duration to 10 minutes, the separation of sister chromatids increased east. This procedure can be coupled with other methods such as antibody staining in order to answer questions like do certain chromatin proteins localized to specific sequences.