3D DNA FISH: A Technique to Locate a Specific Gene on a Chromosome

- 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 canc