3D multicolor DNA FISH enables the visualization of multiple genomic loci within preserved nuclei to ambiguously define their reciprocal interaction and localization at single cell level. 3D multicolor DNA FISH allows the direct investigation of the nuclear architecture. In general, it works in conjunction with chromosome capture-based technologies making the technique an available tool for C data validation within single cells.
Demonstrating the procedure will be Federica Marasca. She's a postdoc in my laboratory. Begin by incubating the nick translation mix in a thermal mixer at 16 degrees Celsius according to the length of the starting DNA material.
At the end of the incubation, check the size of the probes on a 2.2%agarose gel. For each DNA FISH experiment, precipitate the appropriate quantity of probe according to the starting DNA material from which the probes were produced in 150 microliters of double distilled water, 20 micrograms of unlabeled salmon sperm DNA, 3.5 micrograms of species specific Cot-1 DNA, three volumes of 100%ethanol, and a 1/10th volume of three molar sodium acetate for one hour at minus 80 degrees Celsius. At the end of the incubation, centrifuge the sample at maximum speed for one hour at four degrees Celsius.
After discarding the supernatant, wash the pellet two times with 500 microliters of 70%ethanol. After the second wash, resuspend the pellet in two microliters of 100%formamide and shake the probe for 30 minutes at 40 degrees Celsius before adding an equal volume of 4X saline sodium citrate in 20%dextran sulfate. For the fixation pre-treatment and permeabilization of small cells with small nuclei and a low amount of cytoplasm, first add two times 10 to the sixth cells in 200 microliters of PBS directly onto glass coverslips in individual wells of a 24-well plate and allow the cells to settle for 30 minutes at room temperature.
At the end of the incubation, quickly replace the PBS with 300 microliters of freshly prepared 4%paraformaldehyde supplemented with 0.1%Tween 20. After 10 minutes, wash the fixed cells three times in 300 microliters of 0.05%TPBS for five minutes per wash at room temperature. After the last wash, permeabilize the T cells with 300 microliters of 0.5%TPBS for 10 minutes at room temperature before treating the cells with 250 microliters per well of RNAse cocktail diluted 1:100 in PBS for one hour at 37 degrees Celsius.
At the end of the incubation, rinse the cells in 300 microliters of PBS and add 300 microliters of 20%glycerol in PBS to each well with an overnight incubation at four degrees Celsius. The next morning, place the glass on dry ice for 15 to 30 seconds to freeze the cells before gradually thawing the samples at room temperature and soaking them in 300 microliters of 20%glycerol in PBS for 30 seconds. After freeze/thawing the cells three more times as just demonstrated, wash the samples in 300 microliters of 0.5%TPBS for five minutes at room temperature, followed by two washes in 300 microliters of 0.05%TPBS for five minutes per wash at room temperature.
After the last wash, treat the cells with 300 microliters of 0.1 normal hydrochloric acid for 12 minutes at room temperature, followed by two rinses in 300 microliters of saline sodium citrate. Then fix the samples overnight in 300 microliters of 50%formamide in 2X saline sodium citrate at room temperature. For the fixation, pre-treatment and permeabilization of large cells with a high amount of cytoplasm, fix, pre-treat and permeabilize the cells similarly to as demonstrated for human primary T cells and treat the samples with 300 microliters of 0.0025%pepsin in 0.01 normal hydrochloric acid for a few seconds up to five minutes.
Observe the cells under an optical microscope and stop the reaction with two five-minute washes in 300 microliters of 50 millimolar magnesium chloride as soon as the nuclei are free from the cytoplasm but the nucleoli are still visible and intact. For 3D multicolor DNA FISH, denature the hybridization probes at 80 degrees Celsius for five minutes and immediately place the probes on ice. Load the probes onto a clean microscope slide and place one coverslip of cells onto each drop of probe.
Seal the coverslip edges with rubber cement. When the cement has dried completely, denature the samples on a 75 degrees Celsius heating block. After four minutes, hydridize the samples at 37 degrees Celsius overnight in a metallic box floating in a water bath.
The next morning, peel off the rubber cement and with the slides immersed in 2X saline sodium citrate carefully lift off the coverslips. Place each coverslip into individual wells of a six-well plate containing two milliliters of fresh saline sodium citrate per well for two five-minute washes at 37 degrees Celsius with shaking at 90 revolutions per minute. At the end of the incubation, rinse the coverslips briefly in two milliliters of 0.2%Tween 20 in 4X saline sodium citrate.
For 3D multicolor DNA FISH detection, transfer the coverslips into individual wells of a new 24-well plate and block any nonspecific binding in 300 microliters of blocking buffer for 20 minutes at 37 degrees Celsius and 20 revolutions per minute. At the end of the incubation, treat the samples with the appropriate concentration of anti-digoxigenin and/or streptavidin diluted in blocking buffer for 35 minutes in a dark and wet chamber at 37 degrees Celsius. At the end of the incubation, transfer the samples to individual wells of a six-well plate and wash the samples three times in two milliliters of 0.2%Tween 20 and 4X saline sodium citrate for five minutes per wash with shaking at 90 revolutions per minute.
After the last wash, equilibrate the samples in two milliliters of PBS before transferring the coverslips to a new 24-well plate for post-fixing in 300 microliters of 2%formaldehyde in PBS per well for two minutes at room temperature. Wash the fixed cells five times briefly in 300 microliters of PBS, followed by staining with DAPI diluted at one nanogram per milliliter in 300 microliters of PBS for five minutes at room temperature. Wash the coverslips five more times in PBS and mount the samples with an appropriate mounting medium.
Then acquire 3D images with a microscope system. 3D multicolor DNA FISH allows the contemporary visualization of different genomic loci within preserved 3D nuclei. For DNA probe preparation, a probe size of less than 200 base pairs ensures a successful 3D multicolor DNA FISH procedure.
Suboptimal DNA FISH probes produced by nick translation can be partially or over digested. Over digested probes will result in a nonspecific signal due to a loss of specificity in the hybridization and a consequent increase of the background. For human primary T lymphocytes and small cells with small nuclei and a low amount of cytoplasm, a 12-minute 0.1 normal hydrochloric acid treatment is recommended to promote nuclei accessibility to the DNA probes and to preserve the nuclear integrity.
Cytoplasmic pepsin digestion is not needed to obtain a DNA FISH good signal in small cells. For human primary myoblasts and cells that have large nuclei and abundant cytoplasm, however, the pepsinization step is fundamental. A short and suboptimal cytoskeleton pepsinization will hamper the entry of the probe into the nuclei ending in the absence of a DNA FISH signal.
However, if the cells are over pepsinized, the nuclei will not remain intact losing their 3D structure. A successful 3D multicolor DNA FISH will result in the presence of signal within the nuclei. I suggest working with fresh biological material, testing probes before performing the 3D multicolor DNA FISH, preparing fresh solutions and being precise with incubation times and the temperatures.
Remember that formamide and HCL are hazardous substances and should always be used in a chemical fume hood with appropriate disposable materials.
