3D DNA FISH: A Technique to Locate a Specific Gene on a Chromosome
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– Take a functionalized wire with cells attached on its surface and stained by immunofluorescence staining. Wash it with an ice cold saline sodium citrate or SSC solution. Place the wire into a microtube containing fluorescently tagged probes, and close it with a wire stopper. Incubate the microtube in a dark humid chamber in a hybridization oven at 75 degrees Celsius for denaturing the DNA within the cells.
Transfer the chamber into an oven at 37 degrees Celsius for the probes to bind to the complimentary sequence on the DNA. Next, remove the chamber from the oven and take out the wire. Dip the wire into a tube containing SSC solution at the desired temperature. Wash the wire with the SSC solution containing Tween to remove non-specific or partially hybridized probes, and wash again in distilled water.
Dry the wire and incubate the functionalized tip in DAPI solution to stain the cell's nuclei. Rinse the wire in PBS and air-dry. Place the wire under a fluorescence microscope. The probe hybridized with the gene-of-interest appears as a fluorescent spot in the cell's nuclei. In the following protocol, we will perform 3D DNA FISH to determine the ALK gene's translocation in lung cancer cells.
– 3 hours before the DNA FISH protocol, set the hybridization oven to 75 degrees Celsius, and set the dry heat oven at 37 degrees Celsius. Cool the 0.4-fold SSC solution to 4 degrees Celsius. Wash the wire three times in ice cold 0.4-fold SSC solution. Dry the wire in the dark under the fume hood cupboard for 10 minutes.
After 10 minutes, vortex and spin the probe for 5 seconds. Drop 10 microliters of the probe into glass microtubes, then cover them with laboratory film. Wrap the microtubes in dry, absorbent paper placed into a 50 milliliter tube and spin them briefly. Carefully place the dried wire into the microtube and insert the wire stopper. Then, seal the wire stopper with rubber cement.
Place the wire into a dark humid chamber in the hybridization oven for 8 minutes at 75 degrees Celsius to obtain complete DNA denaturation. After 8 minutes, move the humid chamber into a dry heat oven at 37 degrees Celsius overnight. Place a slide staining jar with 0.4-fold SSC solution into a water bath set at 72 degrees Celsius. Check the 0.4-fold SSC solution temperature until it reaches 72 plus or minus 1 degree Celsius.
Next, prepare two glass beakers– one with two-fold SSC plus 0.05% Tween solution, and the second one with distilled water. Remove the humid chamber from the dry heat oven. Use tweezers to carefully remove the rubber cement from the wire stopper and pull the uncoated end of the wire through the end stopper, being careful not to damage the functionalized tip.
Dip the wire into the 0.4-fold SSC solution for 2 minutes at 72 degrees Celsius. Wash the wire in two-fold SSC plus 0.05% Tween solution for 30 seconds at room temperature. Then, wash the wire in distilled water at room temperature. Dry the wire under the fume hood for 10 minutes in the dark. Incubate the functionalized tip of the wire with previously prepared DAPI solution in a 2 milliliter vial. Rinse the wire twice in 1-fold PBS and air-dry the wire.
Position the wire in the wire holder, and carefully insert the functionalized tip through the entry point of the special holder until the tip matches the coupling point. Place the special holder on the microscope stage. Adjust the focus of the microscope using a 20-fold lens. First, coarsely focus on the special support, and then adjust finely on cells that appear bright in the DAPI channel.
