A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

The overall goal of this method is to prepare plant chromosomes suitable for the fluorescence in C two hybridization technique using single and high copy DNA probes. This method can help answer key questions in cytogenetics and genome research, such as detection of chromosome rearrangements, ordering of genetic markers on chromosomes, and comparative analysis between different species. The main advantage of this procedure is that preparation of chromosome spreads becomes fast, easy, and efficient.

Cell suspension can be stored at minus 20 degree up to two months and used for chromosome preparation when needed. We first had this idea of this method when we observed that the quality and the number of suitable chromosomes increased dramatically are human days Germinate 10 to 20 barley seeds on two layers of moist filter paper in a Petri dish under dark conditions for two days at 22 to 24 degrees Celsius after two days. Cut off vigorous roots with lengths of one to two centimeters from the seed by using a razor blade.

Next, prepare ice cold water by placing a 500 milliliter glass bottle containing cold tap water into crushed ice water. Aerate the ice cold water and immerse the root tips for 20 hours to increase the frequency of metaphase cells. Transfer the roots from water to 50 milliliters of ethanol acetic acid fixative to fix them at room temperature for two days after fixation.

Wash 10 to 20 roots with 30 milliliters of ice cold tap water in a 50 milliliter glass speaker for five minutes twice. Transfer the roots one by one into 30 milliliters of citrate buffer using forceps. Wash the roots by shaking the glass speaker for five minutes twice.

Then place the roots on filter paper to remove the liquid completely under a microscope. Cut off the undesired non meristematic tissue using a razor blade. The most critical step of this procedure is to determine the proper digestion time that may vary between different species.

Here up to 20 root tips are incubated in one milliliter of enzyme mixture at 37 degrees Celsius for about 50 minutes. To soften the plant tissue in a watch glass under a microscope, remove the enzyme by pipetting and wash the root tips on ice with five milliliters of 0.01 molar citrate buffer to replace the residual enzyme. Continue to wash the root tips with one milliliter of 96%ethanol twice carefully in the same watch glass.

Then replace the ethanol with 10 to 15 microliters of freshly prepared fixative per root tip. Transfer the root tips together with the fixative into a two milliliter tube and disintegrate the root mar stems with a dissecting needle or forceps. Tap the tube 20 times to resuspend the cells and obtain a cell suspension.

Next place two to three layers of water soaked tissue paper on a hot plate at 50 degrees Celsius. Immerse the microscopic slides in ice, cold tap water in the refrigerator for 30 minutes. Then place the slides on top of the moist tissue paper, pipette seven to 10 microliters of cell suspension, and drop it from a distance of 20 centimeters onto the cooled slide placed on the hot plate.

Follow this with 10 microliters of the acetic acid ethanol mixture on the same place as the cell suspension on the slide, and keep the slide on the hot plate. After two minutes, place the slide on the hot plate without the wet tissue and let it dry for an additional one minute. Check the slides using a face contrast microscope to control the quality of the chromosome spread.

Use the slides either the same day or store them by immersing in 96%Ethanol in a coplin jar at minus 20 degrees Celsius to pretreat the slides before fish. Place the slides in a copin jar containing 50 milliliters of two XSSC for five minutes using forceps. Transfer the slides to a coplan jar containing 50 milliliters of 45%acetic acid for three to 10 minutes.

Next, transfer the slides to a coplan jar containing 50 milliliters of two XSSC for 10 minutes. Then transfer the slides to a coplin jar containing 50 milliliters of 4%formaldehyde, and immerse the slides for 10 minutes to fix the chromosomes. Remove the formaldehyde by rinsing the slides three times for four minutes each in a coplin jar containing 50 milliliters of two XSSC.

Then dehydrate the slides for two minutes in a series of 70%90%and 100%ethanol, and dry the slides in a vertical position. For each slide, prepare a hybridization solution of 20 microliters using 10 microliters of deionized form amide five microliters of four X hybridization buffer, three microliters of the probe and two microliters of D ns free water. Add 20 microliters of hybridization solution per slide and cover with a 24 by 32 millimeter cover slip.

After sealing the cover slip with rubber cement denature slides with probes simultaneously at 80 degrees Celsius for two minutes on a hot plate, transfer the slides to a moist chamber and incubate them at 37 degrees Celsius overnight. Avoiding light, remove the cover slips by rinsing the slides in a coupling jar with two XSSC. Then place the slides in a coplan jar containing 55 to 60 degree Celsius, two XSSC and incubate for 20 minutes.

Next, place the slides in two XSSC in a coplan jar for two minutes at room temperature.Sure. Dehydrate the slides in a coplan jar for two minutes in a series of 70%90%and 100%ethanol.Air. Dry the slides and counterstain with dappy in Antifa mounting medium, avoiding intense light.

Analyze the slides using an epi fluorescence microscope. The selection of filter depends on the fluorochrome used for probe labeling if necessary. Store the slides at four degrees Celsius under dark conditions up to a year.

Mitotic metaphase spreads prepared by the fast air dry dropping chromosome preparation method are shown here. Fish analysis was carried out using both repetitive and single copy sequences. Images were obtained by an epi fluorescence microscope with a set of filters enabling excitation of corresponding fluoro fours and captured by a high sensitivity CCD monochrome camera.

The fish experiment on mitotic metaphase chromosomes of hoard and vulgari using single copy probe and five S-R-D-N-A revealed distinct and high quality signals. Hybridization of CT CTT 10 microsatellite on the chromosomes of hordy and BULBUS resulted in a specific and distinct pattern. The fish experiment on mitotic metaphase chromosomes of sala serial using PSC 119 two probe resulted in specific and high quality signals.

Obvious benefits of this approach are well spread, undamaged, and numerous metaphase chromosomes serving as a perfect prerequisite for successful fish analysis. Once master, this procedure can be done in less than two hours if performed properly. After watching this video, you should have a good understanding on how to prepare the high quality mitotic meta face chromosome space shoot for for fish IDE and former, a height can be extremely dangerous, always to use graphs and film hood when performing these procedures.