Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

1. Slide preparation, sterilization and cell culture

1. Prepare 500 mL of 10% HCl (v/v) and pour into a large beaker.
2. Drop microscope slides individually into the acid and incubate for 1 h at room temperature on a shaker set at 2 x g.
   CAUTION: HCl is corrosive and an irritant. It may cause severe skin burns and eye damage and irritation of the skin, eyes, and respiratory system. Ensure appropriate personal protection is worn including nitrile gloves, eye protection and a laboratory coat.
3. Decant acid from the beaker and wash slides ten times in tap water and then a further ten times in deionized water.
4. Rinse slides in methanol twice and keep in methanol until sterilization by flaming.
   CAUTION: Methanol is a highly flammable liquid and toxic if swallowed, in contact with skin or if inhaled. In addition, methanol may cause damage to organs, is corrosive and an irritant. Adhere to the workplace exposure limits and ensure appropriate personal protection is worn including butyl-rubber gloves, eye protection and a laboratory coat. Where possible handle in a local exhaust ventilation (LEV) fume cupboard.
5. Using metal tongs or long forceps, remove a microscope slide from the beaker containing methanol. Flame over a Bunsen burner to sterilize, and transfer into a rectangular cell culture vessel containing four compartments for slides, located close to the Bunsen burner.
   CAUTION: Flaming allows immediate sterilization of microscope slides prior to use; however, this method does have associated hazards. As methanol is highly flammable it is important that the beaker containing the slides is positioned away from the Bunsen burner. Long tongs or forceps should be used that tightly grip the slides. The methanol level in the beaker should just cover the slides, to both minimize the amount of methanol used and so that only the ends of the forceps/tongs are in contact with the methanol. Always ensure the methanol has evaporated from the forceps or tongs after use and that these have cooled before placing back into the beaker containing the slides and methanol. The beaker should be covered by a piece of aluminum foil to starve the oxygen should the methanol catch fire. Never flame slides within a Class II laminar flow hood where the air is being circulated.
6. Alternatively, perform steps 1.1 to 1.4, but rather than flaming the slides after incubating with methanol, place slides on lint-free tissue to air dry. Once dry, wrap in aluminum foil and place in a sterilizer oven or autoclave.
7. Grow cells in the appropriate medium with serum for at least 48 h at 37 °C in 5% CO₂ until 60-70% confluency is reached. This protocol was performed on an early passage of human dermal fibroblasts (HDFs) and on classical Hutchinson-Gilford progeria syndrome (HGPS) fibroblasts (AG06297) and atypical type 2 HGPS fibroblasts (AG08466). Harvest each cell type and count using a hemocytometer to determine cell density. Seed 1 x 10⁵ cells in 10 mL medium per slide.
   NOTE: The cell density is important as DNA loops from different nuclei may converge if cells become too confluent. Seeding densities may need to be optimized depending on the cell type used as transformed cells may proliferate more quickly, whilst later passage cell cultures may take a longer amount of time to reach the desired confluency.
8. If cells need to be arrested in G₀ to become quiescent, then seed 1 x 10⁵ cells (in 10 mL medium) per slide and leave to grow for 24 h. Wash cells twice with serum-free medium and incubate in standard medium that contains a lower concentration of serum at 0.5% (newborn calf serum, NCS; or fetal bovine serum, FBS) for 7 days.
9. If the proliferative status of the cells is required for the DNA Halo assay, then determine cells that have passed through S-Phase by the incorporation of 5-bromo-2'-deoxy-uridine (BrdU) into the DNA during replication.
   1. Seed cells as normal and grow for 24 h. Remove the culture medium and replace with medium containing BrdU and 5-fluoro-2'-deoxyuridine (3 µg/µL). After a further 24 h, remove the medium, wash the cells once with medium (10% NCS) and then re-feed with fresh medium (10% NCS). Incubate for an additional 24 h and then prepare the slides for the DNA halo assay.

2. Probe preparation

1. Chromosome whole and arm painting probes
   1. Make chromosome probes from the amplification of flow sorted or microdissected chromosomes by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) using the method by Telenius et al.¹⁶. Use DOP-PCR to label chromosome probes with either Biotin-16-dUTP or Digoxigenin-11-dUTP as shown in Table 1. Please check manufacturer's instructions for amplification profile, however, the conditions used for this experiment are shown in Table 2.
   2. Prepare arm or whole chromosome probe by adding together 8 µL of labeled PCR product, 7 µL of Cot-1 DNA, 3 µL of herring sperm, 1/20^th volume of 3 M sodium acetate (pH 5.4) and 2 volumes of 100% ethanol. Incubate the probe solution for a minimum of 30 min at -80 °C.
      NOTE: This method can be used to create single chromosome probes, or multiple chromosome probes if different labels (i.e., Biotin-16-UTP and Digoxigenin-11-dUTP) are used for each chromosome of interest.
      CAUTION: Ethanol is a highly flammable liquid and vapor and may cause serious eye irritation. Keep away from heat, hot surfaces, and ignition sources. Adhere to the workplace exposure limits and ensure appropriate personal protection is worn including butyl-rubber gloves, eye protection and a laboratory coat. Where possible handle in a LEV fume cupboard.
   3. Centrifuge probe solution at 13,700 x g for 15 min at 4 °C and then wash with 70% ethanol. Repeat the centrifugation procedure and discard the supernatant, taking care not to disturb or lose the DNA pellet. Allow the DNA pellet to dry.
   4. Add 12 µL of hybridization buffer (50% formamide, 10% dextran sulphate, 10% 20x saline sodium citrate (SSC; 3 M NaCl, 0.3 M tri-sodium citrate; pH 7.0), 1% (v/v) polyoxyethylene sorbinal monolaurate (Tween-20)) to the DNA pellet. Leave at 37 °C for at least 2 h for the DNA pellet to dissolve into the hybridization buffer.
      ​CAUTION: Formamide is carcinogenic and teratogenic so may cause serious harm to an unborn child. If a woman is pregnant or suspects she is pregnant they should avoid working with formamide. Formamide should be used in a LEV fume hood. Adhere to the workplace exposure limits and ensure appropriate personal protection is worn including butyl-rubber gloves, eye protection and a laboratory coat.
2. DNA isolation from Bacterial Artificial Chromosomes (BACs)
   1. Streak a small portion of the glycerol stock from the BAC clone onto a Luria-Bertani (LB) agar plate (1% (W/V) NaCl; 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1.5% (w/v) Agar Technical, 12.5 µg/mL (w/v) chloramphenicol). Incubate overnight at 37 °C.
   2. Select a single colony from the plate and inoculate 10 mL of LB broth (1% (w/v) NaCl, 1% (w/v) bactotryptone, 0.5% (w/v) yeast extract, 12.5 µg/mL (w/v) chloramphenicol). Leave the solution in a shaking incubator overnight at 37 °C.
      CAUTION: Chloramphenicol is suspected of causing cancer. Handle with care and reduce exposure.
   3. Centrifuge culture at 1,700 x g for 10 min at room temperature.
   4. Discard supernatant and add 300 µL of P1 solution (15 mM Tris (pH 8), 10 mM EDTA, 100 µg/mL RNase A) to the pellet. Vortex vigorously and transfer cells to a 2 mL microcentrifuge tube.
   5. Add 300 µL of P2 solution (0.2 M NaOH, 1% (w/v) sodium dodecyl sulphate (SDS) dropwise to the cells. Invert the closed microcentrifuge tube 5 times and leave at room temperature for a maximum of 5 min.
      CAUTION: Sodium hydroxide is corrosive and may cause severe skin burns and eye damage. It may be corrosive to metals. Handle with care and reduce exposure. Adhere to the workplace exposure limits and ensure appropriate personal protection is worn including nitrile-rubber gloves, eye protection and a laboratory coat. Where possible handle in a LEV fume cupboard.
      ​CAUTION: Sodium dodecyl sulfate is a flammable solid, harmful if swallowed and may cause skin and respiratory irritation. It may also cause serious eye damage. Ensure appropriate personal protection is worn including nitrile-rubber gloves, eye protection and a laboratory coat. Where possible handle in a LEV fume cupboard.
   6. Add 300 µL of P3 (3 M potassium acetate) slowly to the cells and gently mix. Place the microcentrifuge tube on ice for 10 min.
   7. Centrifuge at 8,100 x g for 10 min at 4 °C and transfer the supernatant to a tube containing 800 µL of ice-cold isopropanol. Invert the tube several times and incubate at -20 °C overnight.
      ​CAUTION: Isopropanol is a highly flammable liquid and vapor and may cause serious eye irritation, drowsiness, or dizziness. Keep away from heat, hot surfaces, and ignition sources. Adhere to the workplace exposure limits and ensure appropriate personal protection is worn including nitrile-rubber gloves, eye protection, and a laboratory coat. Where possible handle in a LEV)fume cupboard.
   8. Centrifuge at 8,100 x g for 15 min at 4 °C. Remove the supernatant and transfer to another tube. Add 500 µL of ice-cold 70% ethanol. Invert tube several times and centrifuge at 8,100 x g for 5 min at 4 °C.
   9. Remove the supernatant and air-dry the pellet at room temperature. Once the pellet is dry re-suspend in 40 µL of diethyl pyrocarbonate-treated water (DEPC-treated) and leave at 4 °C overnight. Once fully resuspended remove 5 µL of solution and load on a 1% agarose gel to check for the presence of DNA.
3. Single gene probe preparation of BACs via nick translation
   1. Use commercially available nick translation labeling kits. Alternatively, use the following protocol. See Table 3 for constituents and volumes.
   2. Add the constituents from Table 3 together in a microcentrifuge tube adding the DNA Polymerase I last, gently mix and centrifuge briefly for a few seconds. Incubate the solution at 15 °C for 2 h.
   3. To verify fragment sizes, load 5 µL of the solution onto a 2% agarose gel. The DNA fragment size range should be between 200-600 bp. If the DNA fragment sizes are larger, then continue to incubate the solution for a further 15 min at 15 ˚C and run products on 2% agarose gel.
   4. Stop the nick translation reaction by adding 10 mM EDTA, 0.1% SDS (2.5 µL of 0.5 M EDTA, pH 8.0 in 100 µL and 1 µL of 10% SDS in 100 µL). Heat the solution at 65 °C for 5 min.
   5. To remove unincorporated nucleotides apply BAC probe to a spin column. Commercial spin columns can be purchased, or they can be created using a syringe as follows:
   6. Add 30 g of Sephadex G-50 to 500 mL of column buffer (10 mM Tris-HCl (pH8), 1 mM EDTA, 0.1% SDS). Autoclave the mixture. Also, make 500 mL of column buffer (without Sephadex G-50) and autoclave.
   7. Make spin columns by adding autoclaved glass wool to the bottom of a 1 mL syringe. Fill the 1 mL syringe with Sephadex G-50 in column buffer. Place 1 mL syringe into a 15 mL centrifuge tube that has a microcentrifuge tube with no lid at the bottom. Centrifuge at 1,600 x g for 5 min.
   8. Remove syringe and discard the microcentrifuge tube at the bottom. Add a fresh microcentrifuge tube back into the 15 mL centrifuge tube. Add column buffer (without Sephadex G-50) to the 1 mL syringe and reinsert back into 15 mL centrifuge tube. Centrifuge at 1,600 x g for 5 min. Repeat this step again twice.
   9. Remove syringe and insert into a 15 mL centrifuge tube that contains a new clean microcentrifuge tube. Apply probe to the syringe and collect probe in the microcentrifuge tube.
   10. To precipitate the DNA probe add 5 µL of herring sperm DNA (10 mg/mL), 10 µL of sodium acetate and 2.25 volume of 100% ethanol to the DNA solution. Gently mix the solution and incubate at -80 ˚C for a minimum 1 h. Centrifuge at 13,700 x g for 15 min at 4 °C.
   11. Discard the supernatant and wash the pellet with 200 µL of ice-cold 70 % ethanol for 15 minutes at 4 °C. Remove the supernatant and air dry. Once dry re-suspend pellet in 20 µL of DEPC-treated water at room temperature for several hours or overnight at 4 °C. The probe is now ready to be used or may be stored at -20 °C.
   12. For each slide mix 5 µL probe DNA with 5 µL Cot-1 DNA and dry using a Speed Vac vacuum concentrator. Once the pellet has dried re-suspend in 12 µL of hybridization mix.

3. DNA Halo preparation

1. Remove the square culture dish containing the slides and attached cells from the incubator. Discard medium, label slides using a pencil and place in a Coplin jar containing 50 mL of ice-cold cytoskeleton (CSK) buffer: 100 mM NaCl, 3 mM MgCl₂, 0.3 M sucrose, 10 mM 1, 4-piperazinediethanesulfonic acid (PIPES; pH 7.8), 0.5% (v/v) Triton X-100 made up in deionized water. Incubate for 15 min on ice or at 4 °C.
   CAUTION: Triton X-100 can cause skin irritation and serious eye damage. Handle using appropriate personal protective equipment including nitrile gloves, goggles, and laboratory coat.
2. Discard the CSK buffer and quickly rinse slides in 50 mL of 1x DNA halo buffer (DHB; 140 mM NaCl, 27 mM KCl, 110 mM NaHPO₄, 15 mM KH₂PO₄; pH7.4) three times, i.e., dip slide into Coplin jar containing DHB and remove.
3. Transfer slides to a Coplin jar containing 50 mL of extraction buffer: 2 M NaCl, 10 mM PIPES (pH 6.8), 10 mM ethylenediaminetetraacetic acid (EDTA), 0.1% (w/v) digitonin, 0.05 mM (v/v) spermine, 0.125 mM (v/v) spermidine. Incubate for 4 min at room temperature.
   CAUTION: Digitonin is toxic if swallowed or in contact with skin and is fatal if inhaled. Ensure digitonin is handled in a LEV fume cupboard and wear a laboratory coat, nitrile gloves (double gloved), safety glasses and mask. Both spermine and spermidine may cause severe skin burns and eye damage, whilst EDTA causes serious eye irritation, so handle each chemical with care.
   NOTE: Prepare digitonin separately by dissolving the powder in water at a temperature of 60-70 °C. Add dissolved digitonin to the extraction buffer once cooled. Add spermine, spermidine and digitonin last to the extraction buffer to preserve the biological activity.
4. Incubate slides consecutively in 50 mL of 10x DHB (1.4 M NaCl, 270 mM KCl, 1.1 M NaHPO₄, 150 mM KH₂PO₄; PH7.4), 5x, 2x and 1x DHB for 1 min each.
5. Dip slides (straight-in and straight-out) through a 50 mL sequential ethanol series of 10%, 30%, 70% and 95% (v/v) ethanol.
6. Air dry slides and store at -80 °C until two-dimensional fluorescence in situ hybridization (2D FISH) is performed.

4. Two-dimensional fluorescence in situ hybridization

1. Make 20x SSC: 3 M NaCl, 0.3 M tri-sodium citrate, pH 7.0. This buffer can be autoclaved, stored at room temperature, and diluted as needed.
2. Make 70% (v/v) formamide, 2x SSC pH 7.0 and heat to 70 °C in a water bath.
3. Incubate slides, for 5 min each, through a sequential 50 mL ethanol series of 70, 90 and 100% ethanol.
4. Air dry slides on a warming plate and bake in a 70 °C oven for 5 min.
5. Denature slides by placing in the 70% formamide, 2x SSC solution for 2 min at 70 °C.
   NOTE: The temperature and timing are critical for step 4.5. If the temperature is the DNA will not denature and the probes will not hybridize, and no signal will be obtained from DNA halo FISH.
6. Place the denatured slide in 50 mL of ice-cold 70% ethanol for 5 min and take through an ethanol series of 90%, 95% and 100% at room temperature for 5 min each.
7. Air dry on a warming plate
8. Handle directly labeled total human chromosome probes according to manufacturer's instructions. For these experiments we used human whole chromosome paints 1, 13, 15, 17 and 18. In addition, in this experiment, CCND1 and CTNNA1 gene probes were used.
   NOTE: Both the whole chromosome probes and BAC gene-specific probes were labeled with biotin-11-dUTP and detected by streptavidin conjugated to Cyanine 3 (Cy3). For the chromosome painting probes made by (DOP-PCR) and BAC DNA labeled by nick translation, these will be referred to as DNA probes from this point forward in the protocol and treated as follows.
9. Denature DNA probe (whole chromosome paint or gene-specific probe) at 75 °C for 10 min in a hot-block or water bath.
10. Warm DNA probes at 37 °C for 30 min in a hot-block or water bath before pipetting 10 µL onto the appropriate slide.
    NOTE: This step is important to block repetitive chromosomal sequences. If not performed non-specific signals may be produced in the DNA Halo FISH.
11. Overlay probe with a 21 mm x 21 mm coverslip and seal using rubber cement.
12. Incubate slides for a minimum of 18 h at 37 ˚C in a humidified hybridization chamber.
    NOTE: Humidified hybridization chambers can be made from sandwich boxes that contain several layers of moistened tissue and a raised platform constructed from cut 10 mL plastic pipettes to rest the slides upon. This is covered in aluminum foil to minimize exposure to light.
13. Remove the rubber cement carefully using forceps.
14. Incubate slides in 50 mL 50% (v/v) formamide, 2x SSC, pH 7.0 solution that has been pre-warmed to 45 ˚C for three 5 min incubations.
    NOTE: Allow the coverslip to fall away from the slide in the first incubation in 50% (v/v) formamide, 2x SSC, pH 7.0 solution. This prevents damage to the DNA Halo preparation that could be caused by 'dragging' the coverslip away. The slides can be agitated in the buffer via gripping by forceps to help detach the coverslip.
15. Next, place slides in 50 mL of 0.1x SSC, pH 7.0 solution that has been preheated to 60 ˚C but placed in a 45 ˚C water bath. Incubate for 5 min and replace the buffer two more times with 5 min incubations.
16. Place slides into a Coplin jar containing 50 mL of 4x SSC, pH 7.0 solution at room temperature and incubate for 15 min with three changes of buffer.
17. Apply 100 µL of 4% BSA, 4x SSC solution to each slide and overlay with a piece of paraffin film. Incubate at room temperature for 10 min. This prevents non-specific binding of the antibody.
18. To detect the labeled probe (biotin-16-dUTP), incubate with 100 µL of a 1:200 (made in 1% BSA, 4x SSC) streptavidin-Cy3 for 1 h at room temperature.
    ​NOTE: Follow manufacturer's instructions with dilutions of antibodies and test dilution prior to experiment to ensure a good signal is produced
19. Place slides into a Coplin jar containing 50 mL of 4x SSC (0.5% Tween-20) pH 7.0 solution at room temperature and incubate for 15 min with three changes of buffer. Slides can be mounted at this stage as shown in step 4.21 if immunofluorescence is not required.
20. If the proliferative status of cells made into DNA halos is required, stain with anti-pKi67 antibodies after the FISH steps, prior to mounting or stain for incorporated BrdU.
    1. Wash slides 3 times for 5 min each in 50 mL of 1x phosphate buffered saline (PBS), followed by blocking with 4% NCS in PBS for 1 h at room temperature.
    2. Apply 200 µL of the necessary primary antibody (rabbit anti-human pKi67; mouse anti-BrdU) to the slide, overlay with a strip of paraffin film and incubate at room temperature for 1 h.
    3. Wash slides 3 times for 5 min in 1x PBS and incubate at room temperature for 1 h in 200 µL of fluorochrome-conjugated secondary antibody (pKi67: swine anti-rabbit TRITC; BrdU: donkey anti-mouse Cy3). Perform 3 more 5 min washes with PBS. All dilutions to be made using 1% (v/v) NCS in PBS at the manufacturers' range of suggested dilutions.
21. Mount slides in 20 µL of mountant containing DAPI and overlay with a 22 mm x 50 mm coverslip.

5. Telomere PNA FISH

1. To detect Telomeres, use telomere PNA FISH kit – FITC; perform the procedure with the manufacturer's instructions. The procedure should be executed at room temperature, unless stated otherwise.
2. Immerse slides in tris-buffered saline (TBS, pH 7.5) for 2 min and then place in 3.7% formaldehyde (in TBS; v/v) for exactly 2 min.
   CAUTION: TBS solution contains 10-30% trometamol and 10-30% 2-amino-2-(hydroxymethyl) propane-1,3-diol hydrochloride. This may cause serious eye and skin irritation, so wear protective gloves and goggles/face protection. Use in a well-ventilated area.
3. Wash slides in a Coplin jar twice with TBS for 5 min each.
4. Immerse slides in pre-treatment solution for 10 min and then wash twice with TBS for 5 min per wash.
5. Next, take the slides through an ice-cold ethanol series comprising of 50 mL of 70%, 85% and 95% (v/v) ethanol for 2 min per concentration. Afterwards allow slides to air dry.
6. Apply 10 µL of Telomere PNA Probe/FITC (or Cy3) depending on the choice of fluorescent tag coloration, to each slide and cover overlay with a coverslip. Incubate in a pre-heated oven set at 80 °C for 5 min and then place in the dark for approximately 1 h.
   CAUTION: Telomere PNA Probe/FITC contains 6-100% formamide, which causes serious eye irritation and is teratogenic so may cause serious harm to an unborn child. If a woman is pregnant or suspects she is pregnant they should avoid working with formamide. Formamide should be used in a LEV fume hood and appropriate eye or face protection should be worn.
7. To remove the coverslips, immerse the slides in 'Rinse Solution' for 1 min and then place in the 'Wash Solution' for 5 min at 65 °C.
   ​CAUTION: Wash solution contains 1-5% polyoxyethylene octyl phenyl ether and 1-5% sodium chloride. This is corrosive and can cause serious eye damage. Ensure that goggles or face protection is worn when handling the wash solution.
8. Incubate slides through a 50 mL of ice-cold ethanol series (70%, 85% and 95% (v/v)) for 2 min per concentration and then air dry. Once dry mount slide with mountant containing DAPI and overlay with a coverslip.

6. Image capture and analysis

1. To visualize DNA halos and chromosome territories use an epifluorescence microscope (e.g., Leica DM4000 microscope) capturing images using a HC PL FLUOTAR 100X/1.30 oil objective and DFC365FX camera.
2. Capture grey-scale images and define color for each channel captured to enable pseudocoloring of images. A commercial software was used in this experiment (e.g., LAS AF version 4.5.0 software). The individual color channels were exported as TIFFs.
3. Analyze images using the Java image processing program Fiji ImageJ. Upload image by pressing File and Open.
4. Load separate image channels or split a composite image into separate greyscale channels by clicking Image | Color | Split Channels. Select an image channel and click Image | Adjust and then select Brightness & Contrast. Alter accordingly and repeat with other channels.
5. Create a mask of the residual nucleus by selecting the DAPI-stained channel depicting the nucleus. Click Image | Adjust and then select Threshold. A dialogue box will appear where threshold can be altered, check the Dark background box. Alter until residual nucleus is clear and press Apply and close the dialogue box.
   NOTE: This creates a binary mask based on the pixel intensity, with white pixels showing regions of interest and black pixels showing background. Repeat the same procedure on the probe channel.
6. Use the freehand selection to outline the periphery of the residual nucleus then click Edit and Clear Outside. Overlay the probe channel onto the residual nucleus. This can be done by pressing Image| Color | Merge Channels.
7. To set the measurement scale in ImageJ, draw a line on the scale bar or between the points of two known distances. Go to Analyze and press Set Scale. In the dialogue box add the distance length and click OK. To measure distances, draw a line between the points being measured and click Analyze | Measure. This will transfer the distance values to a data window.
8. Measure the brightest DAPI intensity as this coincides with the center of the nucleus. From this measure the distance from the nuclear center to the furthest chromosome territory edge (CTE). Measure the distance of the nuclear center to the nuclear edge (NE).
9. Ensure that results are portrayed as a CTE/NE ratio. Here the distance from the nuclear center to each furthest chromosome territory edge (CTE) is divided by the distance from the nuclear center to each respective nuclear edge (NE). This should be performed on a minimum of 50 nuclei. This may be depicted as a bar or box chart.
10. For analysis of the telomeres, analyze a minimum of 30 nuclei per dataset. Images can be analyzed using Fiji ImageJ or manually to count the number of telomeres within the residual nucleus and within the DNA halo. BrdU or pKi67 enabled differentiation of proliferating (BrdU/piK67+) and senescent/quiescent (BrdU/pKi67-) nuclei. Data may be depicted in bar charts with error bars corresponding to standard error of the mean (SEM).
11. Use student's t-test (unpaired) to statistically compare the results with p> 0.05 considered significant.