Protocol

Cell Preparation and Multicolor FISH in 3D Preserved Cultured Mammalian Cells

Ludwig-Maximilians University Munich, Department of Biology II, AG Thomas Cremer (Chair of Anthropology and Human Genetics), 82152 Martinsried-Planegg, Germany

¹Corresponding author (Marion.Cremer@lrz.uni-muenchen.de)

INTRODUCTION

Fluorescence in situ hybridization (FISH) on three-dimensional preserved nuclei (3D-FISH) in combination with three-dimensional-microscopy and image reconstruction is an efficient tool to analyze the arrangement of distinct nuclear targets such as entire chromosome territories, chromosomal subregions, or single gene loci on a single-cell level. This protocol focuses on fixation, pretreatments, and 3D-FISH on cultured mammalian cells. It can be applied to a variety of cell types growing adherently or in suspension.

Related information

Figure 1 presents an overview of the methods involved here, as well as in the Preparation of Complex DNA Probe Sets for 3D FISH with up to Six Different Fluorochromes, and FISH on Histological Sections.

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Figure 1. Overview of methods involved in performing FISH on cultured cells and tissue sections. Protocol 4723 is described herein; Protocol 4729 corresponds to FISH on Histological Sections; and Protocol 4730 corresponds to Preparation of Complex DNA Probe Sets for 3D FISH with up to Six Different Fluorochromes.

MATERIALS

Reagents

Antibodies (primary and secondary) or avidin conjugates (see Discussion and Table 1)

Bovine serum albumin (BSA, fraction V) (ICN Biomedicals GmbH)

Cells grown on slides or coverslips (for Steps 1-10) or in suspension (for Steps 11-27)

Counterstain (see Step 52):

DAPI (4',6-Diamidino-2-phenylindole) (5 µg/ml stock in H₂O; store at -20°C)

Propidium iodide (PI, 500 µg/ml stock in H₂O; store at -20°C) (Sigma-Aldrich)

TO-PRO-3 (1 mM stock; store at -20°C) (Invitrogen)

Other DNA counterstains are available (e.g., SYTO16, YOYO, Hoechst dye), but we do not have extensive experience with them in 3D FISH experiments.

Ethanol (80%) (optional; see Step 11)

Fetal calf serum (FCS) (50%) in RPMI (optional; see Step15)

Formamide-SSC solution

Glycerol (20%) in PBS for FISH

Hybridization mixture with dissolved probe (see Preparation of Complex DNA Probe Sets for 3D FISH with up to Six Different Fluorochromes)

HCl (0.1 M) (Merck)

MgCl₂ (50 mM) in PBS for FISH (1X)

Paraformaldehyde (4% or 1%, w/v), freshly prepared in PBS for FISH (pH 7.3)

For fixation, prepare a 4% (w/v) solution of paraformaldehyde in PBS for FISH (use 1X for adherently growing cells or 0.3X for cells grown in suspension). For post-fixation, prepare a 1% (w/v) solution of paraformaldehyde using 1X PBS for FISH. Dissolve by heating and stirring; avoid boiling.

Pepsin (10% stock in H₂O; stored at -20°C) (Sigma-Aldrich) (optional; see Steps 28-35)

PBS for FISH (20X, pH 7.4)

Polylysine (10 mg/ml stock in H₂O; store 1-ml aliquots at -20°C) (Sigma-Aldrich) (for Steps 11-27)

SSC (20X), diluted to 2X and 0.1X

SSC/Tween (0.2% [v/v] Tween 20 in 4X SSC)

Triton X-100 (0.5%, 0.05%, and 0.01%) in 1X PBS for FISH(Merck)

Vectashield mounting medium (Vector)

Equipment

Centrifuge (optional; see Step 14)

Coplin jars or plastic dishes (see Step 1)

Coverslips

For fixing cells in suspension (Steps 11-26), coverslips of various sizes can be used. Thin coverslips are a bit more delicate, but they endure all pretreatment steps (including freezing and thawing in liquid nitrogen), and they provide better image quality for confocal microscopy. An advantage of small coverslips (e.g., 15 x 15 mm) is that they can be placed directly on a microscopic slide for hybridization.

Fluorescence microscope (e.g., Leica SP2 confocal microscope)

Visualization and acquisition of three-dimensional image stacks requires a fluorescence microscope (wide-field epifluorescence with motorized Z-stage, confocal microscope, microscope with Nipkow disc, or structured illumination) with an appropriate filter set combination for exciting and emitting different fluorochromes. The set of filters available in a lab will determine the experimental setup (see Discussion). For details, see Walter et al. (2006).

Heating block at 75°C

Incubator at 37°C, 5% CO₂

Liquid nitrogen

Metal boxes (serve as dark, moist chambers when floating in water baths at 37°C)

Nail polish (colorless)

Parafilm (optional; see Step 11)

Paper towels

Pasteur pipette, plastic (optional; see Step 2)

Petri dish (optional, see Step 11)

Rubber cement

Shaker

Slides

Water baths (shaking) at 37°C, 60°C

METHOD

For adherently growing cells, begin with Step 1. For cells growing in suspension, begin with Step 11.

Fixation of Adherently Growing Cells for 3D-FISH (~2.5 h)

1. Rinse slide or coverslip with cells briefly in two to three changes of PBS for FISH (hereafter, referred to simply as PBS) at 37°C.
To avoid drying of cells, perform steps (especially washings and changing incubation medium) by quickly transferring the slides from one Coplin jar (or plastic dish) to the next.

2. Fix the cells in 4% paraformaldehyde (freshly made) for 10 minutes at room temperature (RT).
(Optional) During the last minute, add five drops of 0.5% Triton X-100 for every 100 ml of fixation solution using a plastic Pasteur pipette.

3. Wash the cells three times for 3 minutes each with 0.01% Triton X-100 at RT.

4. Incubate the cells in 0.5% Triton X-100 for 5-15 minutes at RT.

5. Incubate the cells in 20% glycerol for a minimum of 60 minutes (preferably overnight) at RT.

6. Freeze the cells by dipping the slide or coverslip into liquid nitrogen (for ~30 sec) and thaw on a paper towel. As soon as the frozen layer disappears, put the slide or coverslip back into 20% glycerol. Repeat four times.

7. Wash the cells three times for 10 minutes each in PBS.

8. Incubate the cells in 0.1 M HCl for 5 minutes at RT.
The time may be extended up to 10 minutes for slides/coverslips with densely grown cells and/or with nuclei embedded in a voluminous cytoplasm. The concentration of HCl should not be varied.

9. Incubate the cells twice for 3 minutes in 2X SSC.

10. Incubate the cells in the formamide-SSC solution for at least 1 hour at RT (preferably overnight) before proceeding with the optional pepsin digestion (Step 28) or hybridization (Step 36).
For long-term storage, keep slides in formamide-SSC at 4°C. Longer storage than 3-4 months may result in deterioration of the nuclear morphology after the denaturation step of 3D-FISH.

Fixation of Cells Growing in Suspension for 3D-FISH (~4.5 h)

11. For polylysine-coated coverslips, dilute 10 mg/ml polylysine stock solution to 1 mg/ml in H₂O. Put ~150 µl of the 1 mg/ml solution on a piece of Parafilm or in a Petri dish. Place a dry coverslip (rinsed in 80% ethanol) on the drop and incubate for 1 hour.
Microscope slides can be used instead of coverslips. Place ~200 µl of polylysine on a slide, covering an area of about 20 x 20 mm. Mark the coated area.

12. Rinse the coverslips carefully in H₂O and air dry.

13. To seed the cells, apply ~1 ml of culture medium containing ~1 x 10⁵ - 1 x 10⁶ cells per 20 x 20-mm coverslip.
For peripheral blood cells, isolate the desired cell type according to the appropriate method. Cells obtained from 1 ml of peripheral blood seeded on a 20 x 20-mm area should yield sufficient cell density for hybridization.

14. Centrifuge the cell suspension at 1000 rpm for 10 minutes.

15. Discard the supernatant and resuspend the pellet in 50% FCS (in RPMI). To increase cell density on the slide, use about one-fourth of the initial volume for resuspension.

16. Place about 200 µl of the cell suspension on a polylysine-coated coverslip, and incubate the coverslip for 1 hour at 37°C in an incubator containing 5% CO₂.

17. Check attachment of cells under the microscope; briefly drain off the medium.
To avoid drying of the cells, perform steps (especially washings, changing incubation medium) by quickly transferring the slides from one Coplin jar (or six-well plastic dish for small coverslips) to the next.

18. Incubate the cells in 0.3X PBS for 40 seconds.
This prevents the shrinkage of spherical cells that are otherwise prone to collapse during the following fixation. However, adhere strictly to the time indicated, otherwise the nuclei will increase in size.

19. Fix the cells in 4% paraformaldehyde for 10 minutes at RT.

20. Wash the cells three times for 5 minutes each in 0.05% Triton X-100 at RT.

21. Incubate the cells in 0.5% Triton X-100 for 20 minutes at RT.

22. Transfer the coverslips to 20% glycerol, and incubate them for at least 30 minutes at RT (or preferably, overnight at 4°C).

23. Freeze the cells by dipping the coverslip into liquid nitrogen (for ~30 sec), and then thaw on a piece of paper towel. As soon as the frozen layer disappears, put the coverslip back into 20% glycerol. Repeat this four times.

24. Wash the cells three times for 5 minutes each in 0.05% Triton X-100.

25. Incubate the cells in 0.1 M HCl for 5 minutes.
See the note to Step 8.

26. Wash the cells twice for 1 minute each in 2X SSC.

27. Incubate the cells in the formamide-SSC solution for at least 1 hour (preferably overnight) at RT before proceeding with the optional pepsin digestion (Step 28) or hybridization (Step 36).
For-long term storage, keep slides in formamide-SSC at 4°C. Slides may be stored for at least 3-4 months. Longer storage may result in a deterioration of the nuclear morphology.

Treatment with Pepsin (Optional; ~45 min)

Pepsin incubation is required for most cell types with a large cytoplasm and/or cultures with a high cell density. Test a sample without pepsinization first to determine whether there is sufficient probe penetration. If pepsinization is necessary, monitor the treatment under the microscope and stop as soon as reduction of cytoplasm is visible.

28. Incubate the slides (kept in formamide-SSC) in 2X SSC for 2 minutes at RT.

29. Incubate the slides in PBS for 3 minutes at RT.

30. Prepare a pepsin working solution (0.005% in 0.01 M HCl), and incubate the slides in it as follows:

i. Add 50 µl of 10% pepsin stock solution to 10 ml of 0.1 M HCl and adjust the volume to 100 ml with H₂O.

ii. Warm the pepsin working solution to 37°C.

iii. Pour the pepsin working solution into a Coplin jar, and incubate the slides in it for 3-5 minutes.

31. To inactivate the pepsin, incubate the slides twice for 5 minutes each in 50 mM MgCl₂ at RT.

32. Post-fix the cells in 1% paraformaldehyde for 10 minutes at RT.

33. Wash the cells in PBS for 5 minutes at RT.

34. Wash the cells twice for 5 minutes each in 2X SSC.

35. Return the slides to the formamide-SSC solution for at least 30 minutes.

Probe Denaturation (~1 h) and Hybridization (~1-3 d)

Cellular and probe DNA can be denatured simultaneously, even with probes that require a high excess of Cot1-DNA. Simultaneous denaturation is quick, simple, and optimal for retention of three-dimensional morphology.

36. Take a slide with cells out of the formamide-SSC and quickly drain off the excess fluid.
If cells are grown on large coverslips (e.g., >18 x 18 mm), cut the coverslip with the cells (without letting them dry out!) to the appropriate size.

37. Place the hybridization mixture with dissolved probe on a coverslip (e.g., 6-8 µl probe per 18 x 18-mm coverslip), and cover the target area of the slide (cells) with the coverslip (probe).
For cells grown on coverslips, place the hybridization mix directly on a microscope slide and cover with a coverslip (with cells facing the drop).

38. Wipe off the excess fluid around the coverslip and seal with rubber cement. Let the rubber cement dry completely.
Protect from light during this and all subsequent steps if fluorochrome-labeled probes are used.

39. Place slides on a heating block at 75°C for 2 minutes to denature cellular and probe DNA.
Do not vary this time or temperature.

40. Perform hybridization in a metal box floating in a 37°C water bath at least overnight (preferably for 2-3 d).

Washing and Detection (~1-5 h)

If fluorochrome-labeled probes are used, perform all steps under light protection.

41. After hybridization, peel off the rubber cement, gently remove the coverslip, and transfer the cells to 2X SSC.
If the coverslip cannot be stripped off easily, incubate briefly in 2X SSC and try again.

42. Wash the cells three times for 5 minutes each in 2X SSC at 37°C with shaking.

43. Wash the cells three times for 5 minutes each in 0.1X SSC at 60°C (stringent washes) with shaking.
If only fluorochrome-labeled probes are used directly, immediately proceed to DNA counterstaining (Step 52).

44. Rinse the cells briefly in SSC/Tween.

45. Prepare a blocking solution of 4% (w/v) BSA in SSC/Tween by adding 2 g of BSA to 50 ml of SSC/Tween. Incubate the cells in the blocking solution for 10-15 minutes at 37°C.

46. Prepare a solution of SSC/Tween containing 1% BSA and dilute the required antibodies or avidin conjugates to the appropriate working concentration in this solution.

47. Incubate the cells with the primary antibody (first layer) in a dark moist chamber for 45 minutes at 37°C.

48. Wash three times for 3 minutes each in SSC/Tween with shaking.

49. Incubate with the appropriate concentration of secondary antibody (second layer) in a dark moist chamber for 45 minutes at 37°C.

50. Wash three times for 3 minutes each in SSC/Tween with shaking.

51. (Optional) Add further layers as needed.

52. For DNA counterstaining, options include:

i. DAPI: From stock, prepare a fresh solution of 0.05 µg/ml DAPI in 2X SSC. Incubate cells in this solution for 2-5 minutes (or longer) at RT.

ii. TO-PRO-3: From stock, prepare a fresh solution of 1 µM TO-PRO-3 in 2X SSC. Incubate cells in this solution for 5-10 minutes at RT.
Longer incubation may lead to strong overstaining.

iii. PI: From stock, prepare a solution of 25 µg/ml PI in 2X SSC. Incubate cells in this solution for 2-5 minutes at RT.

53. Wash briefly in SSC/Tween.

54. Mount hybridized areas in Vectashield.

55. Seal coverslips with colorless nail polish and examine under a microscope.
See Troubleshooting.

56. Store in the dark at 4°C.
FISH signals should be stable for at least 1 year, but counterstain, especially TO-PRO-3, tends to fade after a couple of weeks or months.

TROUBLESHOOTING

Problem: Nuclei are shrunken or frayed after hybridization.

[Step 55]

Solution: Consider the following:

1. Make sure that the cells are not drying out.

2. Reduce the denaturation time and/or temperature.

3. Reduce or eliminate the pepsin treatment.

Problem: There is weak hybridization efficiency of the probes.

[Step 55]

Solution: Test the probe on a metaphase slide. If hybridization signals are weak on metaphase chromosomes, check the detection scheme and probe quality with regard to the appropriate probe length and amount of probe (see Preparation of Complex DNA Probe Sets for 3D FISH with up to Six Different Fluorochromes). If hybridization on metaphases gives good results, add or increase pepsin pretreatment of the three-dimensional-preserved cells.

Problem: There is strong nonspecific background staining.

[Step 55]

Solution: Try the following:

1. Increase the Cot-1 DNA concentration in the probe.

2. Check the concentration of the antibodies used in detection.

3. Check the probe length.

DISCUSSION

The method described here is somewhat delicate, as there are two conflicting goals: preserving nuclear morphology in fixed cells and making chromatin accessible for probe penetration. Paraformaldehyde is likely the most gentle of current fixatives. Structural preservation of chromatin has been demonstrated throughout the whole 3D-FISH procedure, down to individual replication foci (Solovei et al. 2002a,b) that likely represent ~1-Mb chromatin domains (Cremer et al. 2006). Pretreatment steps should be adjusted to the cell type and requirements of hybridization probes in order to balance the preservation of nuclear morphology and hybridization efficiency. Triton X-100 and repeated freezing in liquid nitrogen after incubation in glycerol help make nuclear DNA accessible to FISH probes without significantly affecting the three-dimensional chromatin architecture. Additional deproteinization is necessary when single-copy DNA sequences are targeted, either by incubation in HCl and/or digestion with pepsin.

The choice of detection scheme depends on the number of haptens and fluorochromes used for probe labeling, the number of antibody layers required to obtain a sufficient signal, the color of nuclear counterstain, and most importantly, the microscope setup available. Avoid cross-reactions between antibodies used for the detection of different haptens. Combinations of up to six different fluorochromes can be used successfully, with a Leica SP2 confocal microscope for visualization. A five-color detection scheme works best using Alexa 488 (or FITC) in combination with Cy3 (or TAMRA), Texas Red, Cy5, and DAPI as the DNA counterstain. One or two more fluorochromes can be added (e.g., Alexa 514 and/or Alexa 633), but linear unmixing ("spectral unmixing") of fluorochromes is required after acquisition of image stacks (Walter et al. 2006). Commercially available fluorochrome-conjugated antibodies from the established companies work well. Table 1 summarizes a detection scheme for the combined use of different fluorochromes. For five-color experiments, omit Alexa 514; in three-color experiments, omit Alexa 514 and Texas Red. If the confocal microscope is not equipped with a UV laser, counterstain the slide with TO-PRO-3 or PI. Since PI partially emits in the same spectral range as Cy3 and Texas Red, select fluorochromes to label the probes accordingly. Figure 2 shows an example of a six-color 3D-FISH experiment.

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Figure 2. Six-color 3D-FISH on nuclei of human fibroblasts. Maximum intensity projection of a confocal image stack with six color channels is shown as original images (upper row) and after linear color unmixing (bottom row) using the software of Leica SP2. The FITC channel delineates the territories of chromosome 12, Alexa514 the territories of chromosome 11, and TAMRA the territories of chromosomes 17, 19, and 20. Texas Red delineates a bacterial artificial chromosome (BAC) contig of chromosome 11, and Cy5 a BAC pool covering different regions of chromosome 12. White arrows point at the image regions generated due to "leakage" of some fluorochromes to the neighboring channels, e.g., Alexa514 to the FITC channel (and vice versa), or TAMRA to the Texas Red channel.

Limitations of the technique relate not only to the number of fluorochromes that may be studied in one sample, but to the resolution of current microscopes (200-300 nm for lateral resolution and 600-800 nm for axial resolution). Systems with spatially modulated illumination with a higher resolution both in the axial and lateral directions (Gustafsson 2005) should prove helpful.

ACKNOWLEDGMENTS

The protocols were developed as part of our ongoing studies supported by grants from the Deutsche Forschungsgemeinschaft (Cr 59/20-1-3, Cr 59-26-1/2, and Mu 1850/2-1), the Bundesministerium für Bildung und Forschung NGFN II- EP (0313377A), the Wilhelm-Sanderstiftung (2001.079.2), and the EU 3D- Genome Project (ESF FP6-503441).

REFERENCES

Cremer, T., Cremer, M., Dietzel, S., Muller, S., Solovei, I., and Fakan, S. 2006. Chromosome territories--a functional nuclear landscape. Curr. Opin. Cell Biol 18: 307–316.[Medline]

Gustafsson, M.G. 2005. Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. 102: 13081–13086.[Abstract/Free Full Text]

Müller, S., Neusser, M., Köhler, D., and Cremer, M. 2007. Preparation of complex DNA probe sets for 3D FISH with up to six different fluorochromes. CSH Protocols doi:101101/pdb.prot4730.

Solovei, I., Grasser, F., and Lanctôt, C. 2007. FISH on histological sections. CSH Protocols doi:101101/pdb.prot4729.

Solovei, I., Walter, J., Cremer, M., Habermann, F., Schermelleh, L., and Cremer, T. 2002a. FISH on three-dimensionally preserved nuclei. In FISH: A practical approach (eds. B. Beatty et al.), pp. 119–157. Oxford University Press, Oxford, UK.

Solovei, I., Cavallo, A., Schermelleh, L., Jaunin, F., Scasselati, C., Cmarko, D., Cremer, C., Fakan, S., and Cremer, T. 2002b. Spatial preservation of nuclear chromatin architecture during three-dimensional fluorescence in situ hybridization (3D-FISH). Exp. Cell Res 276: 10–23.[Medline]

Walter, J., Joffe, B., Bolzer, A., Albiez, H., Benedetti, P.A., Muller, S., Speicher, M.R., Cremer, T., Cremer, M., and Solovei, I. 2006. Towards many colors in FISH on 3D-preserved interphase nuclei. Cytogenet. Genome Res 114: 367–78.[Medline]