Protocol

FISH on Histological Sections

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

¹Corresponding author (Irina.Solovei@lrz.uni-muenchen.de)

INTRODUCTION

Introduction: Here we describe fluorescence in situ hybridization (FISH) of DNA probes to histological sections, which allows the visualization of specific DNA targets (chromosome territories and their subregions) in the context of functional tissue organization. Separate protocols are provided for hybridization using paraffin-embedded tissue sections and for hybridization using vibratome or frozen sections. Pretreatment with heat or protease is necessary to allow unmasking of the target DNA and efficient penetration of reagents in the nuclei. Because the goal of the technique is to obtain data on the native 3D structure of the genome, close attention is paid to the preservation of nuclear morphology.

Related information

Figure 1 presents an overview of the methods involved here, as well as in Cell Preparation and Multicolor FISH in 3D Preserved Cultured Mammalian Cells and Preparation of Complex DNA Probe Sets for 3D FISH with up to Six Different Fluorochromes. See Solovei et al. (2002) for more information.

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

Materials

Reagents

Acetone (prechilled) (Merck) (optional; see Step 28)

Antibodies (primary and secondary) and/or avidin conjugates

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

DAPI (4',6-Diamidino-2-phenylindole; 0.2 mg/ml stock in H₂O, sterile filtered) (Sigma-Aldrich)

On the day of the experiment, prepare 0.05 µg/ml DAPI (from stock) in SSC/Tween.

Ethanol (100%, 95%, 70%, 50%, 30%) (Merck)

Formamide-SSC solution

For Step 14.iii, prepare the formamide-SSC solution with 70% formamide.

For Steps 10 and 30, prepare the formamide-SSC solution with 50% formamide.

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

Formaldehyde (2% or 4%, w/v) prepared in PBS

For fixation, prepare a 4% (w/v) solution; for post-fixation (needed only for FISH on Vibratome Sections and Cryosections), prepare a 2% (w/v) solution. Dissolve by heating to 60°C-70°C and stirring. Always prepare fresh.

PBS (20X, pH 7.4), diluted to 1X

Pepsin (10% stock in H₂O) (Sigma-Aldrich) (optional; see Unmasking DNA section for FISH on Paraffin-Embedded Tissue Sections)

On the day of the experiment, prepare a pepsin working solution (0.005% in 0.01 M HCl) by adding 50 µl of 10% pepsin stock solution to 10 ml of 0.1 M HCl. Add H₂O to make 100 ml and warm to 37°C.

Propidium iodide (PI) (Sigma-Aldrich) (optional; see Steps 18, 38)

Proteinase K (Roche) (optional; see Unmasking DNA section for FISH on Paraffin-Embedded Tissue Sections)

Prepare 100 µg/ml proteinase K in 25 mM Tris-Cl (pH 7.4) containing 5 mM EDTA.

Saponin (SERVA) (optional; see Step 37)

Sodium citrate buffer (10 mM, pH 6.0) (Merck) (for FISH on Vibratome Sections and Cryosections only)

Sodium isothiocyanate (1 M, freshly prepared) (Sigma-Aldrich) (optional; see Step 3)

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

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

Tissue sections (paraffin-embedded, vibratome or cryosections)

Vibratome sections (for Step 21) should be cut to a thickness of 50 µm and stored in PBS containing 0.04% sodium azide at 4°C.

Cryosections (for Step 22) should be cut to a thickness of 15-30 µm, placed on SuperFrostPlus slides, immediately frozen on dry ice, and stored at –80°C.

TO-PRO-3 (Invitrogen) (optional; see Steps 18, 38)

Tris-Cl (25 mM, pH 7.4) containing EDTA (5 mM) (optional; see Step 6)

Triton X-100 (Merck) (optional; see Step 37)

Triton X-100 (0.5%, v/v) in PBS for FISH (optional; see Step 24)

Vectashield mounting medium (Vector)

Xylene (Merck) (for FISH on Paraffin-Embedded Tissue Sections only)

Equipment

Brushes (thin) (for FISH on Vibratome Sections)

Coplin jars (glass and plastic)

Coverslips

Glass chambers for mounting of probes (Fig. 2 ) (optional; see Mounting of Probes section under FISH on Vibratome Sections and Cryosections)

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Figure 2. Schematic representation of glass chamber used for hybridization. To prepare glass chambers, cut glass strips from a coverslip (0.17-mm thick) using a diamond cutter, and glue them parallel on two borders of an intact coverslip (e.g., 12 x 12 mm) using nail polish. The section (purple) should first be covered by the glass chamber before filling it with hybridization mixture (blue) from one of the open sides.

Heating block preset to 80°C and/or 85°C (see Steps 13, 14.i, and 34)

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

Microscope slides and coverslips

Microscopes (wide-field epifluorescence and confocal with appropriate filter set combinations; see Walter et al. [2006])

Microwave oven (set at 700 W) (for FISH on Vibratome Sections and Cryosections)

Nail polish

Oven preset to 37°C (optional; see Step 1)

Plastic container (microwave-safe) with plastic slide holder (for FISH on Vibratome Sections and Cryosections; see Step 26)

Rubber cement

SuperFrostPlus slides

Tissues (e.g., Kimwipes)

Water baths (preferably with shaking; preset to 37°C and 60°C)

Additional water baths preset to 45°C, 75°C, and 80°C are required for FISH on Paraffin-Embedded Tissue Sections.

Methods

Follow Steps 1-20 for paraffin-embedded tissue sections or Steps 21-40 for vibratome sections and cryosections.

FISH on Paraffin-Embedded Tissue Sections

1. Deparaffinize sections by immersing in three changes of 100% xylene for 10 minutes each at 45°C.
If the slides have been stored at 4°C, make sure that they are dry before immersing in xylene in order to prevent condensed H₂O from contaminating the xylene solution. If necessary, incubate the slides for 30 minutes in a dry oven (37°C) after removal from storage.

2. Rehydrate the sections in ethanol as follows:

i. Immerse the sections in 100% ethanol, twice for 15 minutes each.

ii. Immerse the sections in 95% ethanol for 5 minutes.

iii. Immerse the sections in 70% ethanol for 5 minutes.

iv. Immerse the sections in 50% ethanol for 5 minutes.

v. Immerse the sections in H₂O for 5 minutes.

Unmasking DNA
To unmask DNA, treat sections either with a denaturing chemical and pepsin (Steps 3-5) or with proteinase K (Steps 6-10). The choice and duration of treatment should be determined empirically.

3. To unmask DNA by treating with pepsin, permeabilize the tissue by incubating the sections with 1 M sodium isothiocyanate (freshly prepared) for 30 minutes in a water bath at 80°C. Rinse briefly in H₂O.

4. Treat the sections with the pepsin working solution for 30 minutes at 37°C. Rinse in H₂O.

5. Dehydrate the slides as follows:

i. Immerse in 70% ethanol for 10 minutes.
For long-term storage, slides can be kept in 70% ethanol at 4°C.

ii. Immerse in 100% ethanol, twice for 10 minutes each.

iii. Air dry at room temperature (RT), and then proceed to denature cellular DNA and probe simultaneously (Steps 11-13) or separately (Step 14).

6. To unmask DNA by treating with proteinase K, incubate the slides in 25 mM Tris-Cl (pH 7.4) containing 5 mM EDTA for 5 minutes at RT.

7. Digest the sections with 100 µg/ml proteinase K for 10 minutes at RT. Rinse in 1X PBS (hereafter referred to as PBS).

8. Fix in 4% formaldehyde for 10 minutes at RT.

9. Wash twice with PBS for 5 minutes each.

10. Equilibrate the slides in formamide-SSC for at least 4 hours, and then proceed to denature cellular DNA and probe simultaneously (Steps 11-13) or separately (Step 14).
Slides can be stored in formamide-SSC for up to 2-4 months at 4°C.

Denaturation of Cellular and Probe DNA
Denature cellular DNA and probe simultaneously (Steps 11-13) or separately (Step 14). Simultaneous denaturation of cellular and probe DNA is quick, simple, and optimal for retention of tissue morphology and is, therefore, the recommended approach, even in the case of probes that require a high excess of Cot-1 DNA.

11. To denature cellular DNA and probe simultaneously, load the hybridization mixture with dissolved probe on the section. Cover the probe with a coverslip. Avoid making air bubbles. Seal the coverslip with rubber cement and allow it to dry completely at RT.
If fluorophore-labeled probes are used, protect the slides from light throughout the remainder of the protocol in order to avoid probe bleaching.

12. Incubate the slides with mounted probe for 1-2 hours at 37°C.
Incubation time can be extended to 12-20 hours with no noticeable increase in background.

13. Simultaneously denature the cellular DNA and probe by incubating the slides with mounted probe for 5 minutes on a heating block set at 85°C. Continue with Step 15.

14. Denature the cellular DNA and probe separately as follows:

i. Denature the probe for 5 minutes at 80°C.

ii. Let the probe preanneal (to the Cot-1 DNA) for 30 minutes at 37°C.

iii. Incubate the slide for 5 minutes at 75°C in a Coplin jar filled with prewarmed formamide-SSC solution (prepared with 70% formamide).

iv. Pull the slide out of the Coplin jar and remove excess formamide. Load the preannealed probe, cover with a coverslip, and seal with rubber cement. Proceed to Step 15.
Perform this step quickly to prevent the sections from drying out.
If fluorophore-labeled probes are used, protect the slide from light throughout the remainder of the protocol to avoid bleaching the probe.

Hybridization and Detection

15. Hybridize by incubating the slides in humid dark chambers (e.g., metal boxes floating in a water bath) for 2-3 days at 37°C.

16. Peel off the rubber cement, flick off the coverslips, and quickly transfer the slides into 2X SSC.

i. Wash three times in 2X SSC (preferably with shaking) for 10 minutes each at 37°C.

ii. If appropriate, wash at high stringency in 0.1X SSC for 10 minutes at 60°C.
If fluorophore-labeled probes were used, proceed directly to Step 18. In the case of hapten-labeled probes, continue with Step 17.

17. For hapten-labeled probes, perform detection as follows:

i. 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 slides in this solution for 15 minutes.

ii. Dilute the primary and secondary antibodies (and/or conjugated avidin) in the blocking solution.

iii. Incubate the sections with antibodies (and/or conjugated avidin) under coverslips in dark humid containers for 1 hour at 37°C.

iv. After each incubation with antibody (and/or conjugated avidin), wash three times with SSC/Tween (warmed to 37°C) for 5 minutes each.

18. Counterstain by incubating slides in 0.05 µg/ml DAPI (prepared in SSC/Tween) for 10 minutes. Rinse briefly in 2X SSC.
Alternatively, counterstain nuclei with 1 µM TO-PRO-3 for 5 minutes (far-red fluorescence) or with 25 µg/ml PI for 15 minutes (red fluorescence), both in SSC/Tween. Note that PI also stains RNA.

19. Place a drop of Vectashield on the top of each section and cover with a coverslip. Avoid making air bubbles. Gently remove excess Vectashield with soft tissue and seal with nail polish.

20. When the nail polish is dry, examine the preparation under the microscope.

FISH on Vibratome Sections and Cryosections

Prepare vibratome sections or cryosections by following Steps 21 or 22, respectively, and then proceed to Step 23.

21. Prepare vibratome sections (stored in PBS with 0.04% sodium azide at 4°C) as follows:

i. Incubate vibratome sections in H₂O for 5 minutes.

ii. Dehydrate the sections in an ethanol series: once in 30% and 50% ethanol for 10 minutes each, and then twice in 70% for 30 minutes each.

iii. Transfer the sections to SuperFrostPlus slides in a drop of 70% ethanol, spread with thin brushes.

iv. Air-dry the slides at RT for 1-2 days.

22. For cryosections (stored attached to SuperFrostPlus slides at –80ºC), thaw the slides at RT on the day before hybridization, and leave them to dry overnight.

23. Rehydrate sections in PBS for 15 minutes.

24. (optional) Permeabilize the sections by incubating in 0.5% (v/v) Triton X-100 in PBS for 20 minutes at RT.
This step can increase probe/antibody penetration in very dense tissues.

25. Put the slides in a plastic slide holder, and place the slide holder in a microwave-safe plastic container filled with 200 ml of 10 mM sodium citrate buffer (pH 6.0). Incubate the slides for 10 minutes at RT.
The slides should be completely covered with liquid.

26. Unmask the DNA as follows:
This is the most critical step of the protocol. The optimal number and duration of heating pulses should be determined empirically for each tissue and probe set. The parameters given here have been used for the hybridization of bacterial artificial chromosome (BAC) probes to 20-µm cryosections of midgestation mouse embryos fixed for 16-20 hours using 4% formaldehyde.

i. Put the container in a microwave set at 700 W, and heat for 2.5 minutes or until first signs of boiling.
The temperature of the citrate solution varies between ~70°C (cooling period) and ~90°C (microwave heating). In order to preserve nuclear morphology, do not let the solution boil.

ii. Cool down for 2 minutes.

iii. Resume heating for 15-25 seconds, until first signs of boiling.

iv. Repeat Steps 26.ii and 26.iii seven times.
This is the most critical step of the protocol. The optimal number and duration of heating pulses should be determined empirically for each tissue and probe set. The parameters given here have been used for the hybridization of bacterial artificial chromosome (BAC) probes to 20-µm cryosections of midgestation mouse embryos fixed for 16-20 hours using 4% paraformaldehyde.

27. Transfer the slides to 2X SSC.

28. (optional) Permeabilize the sections by incubating the slides in prechilled 100% acetone for 5 minutes at –20°C. Wash three times with 2X SSC for 5 minutes each at RT.
This step can increase probe/antibody penetration in dense tissues.

29. If desired, immunostain at this point. After immunostaining, post-fix sections with 2% formaldehyde for 10 minutes at RT.
If immunostaining is performed, consider Step 26 to be the antigen retrieval treatment; it should be adjusted according to the antibody that is being used.

30. Incubate the slides in formamide-SSC solution for at least 4 hours.
Slides can be stored in formamide-SSC for up to 2-4 months at 4°C.

Mounting of Probes
Mount probe under coverslips (Step 31) or, preferably, under small glass chambers specifically designed for DNA hybridization on relatively thick tissue sections (Step 32; see Fig. 2).

31. Mount probe using coverslips as follows:

i. Take slides out of formamide-SSC and remove excess liquid around the sections using tissue.

ii. Load the hybridization mixture with dissolved probe on the section.
If fluorophore-labeled probes are used, protect the slide from light throughout the remainder of the protocol in order to avoid probe bleaching.

iii. Cover the probe with a coverslip. Avoid making air bubbles.

iv. Seal with rubber cement and leave to completely dry at RT. Proceed to Step 33.

32. Mount the probe using glass chambers as follows (see Fig. 2 for preparation of glass chambers):

i. Take slides out of formamide-SSC, and remove excess liquid around the sections using tissue.

ii. Cover the section with the glass chamber.

iii. Fill the glass chamber with probe by capillary action.
If fluorophore-labeled probes are used, protect the slides from light throughout the remainder of the protocol in order to avoid bleaching of the probe.

iv. Seal the chamber with rubber cement and leave to completely dry at RT. Continue with Step 33.

Hybridization and Detection

33. Prehybridize by incubating the slides with mounted probe for 1-2 hours at 37°C.
Incubation time can be extended to 12-20 hours with no noticeable increase in background.

34. Denature the cellular DNA and probe simultaneously by incubating the slides with a mounted probe on a heating block for 5 minutes at 80°C.

35. Hybridize by incubating the slides in humid dark chambers (e.g., metal boxes floating in a water bath) for 2-3 days at 37°C.

36. Perform post-hybridization washings:

i. Peel off rubber cement, flick off the coverslips, and quickly transfer the slides to 2X SSC.

ii. Wash three times for 5 minutes each in 2X SSC buffer (preferably with shaking) at 37°C.

iii. If appropriate, wash three times for 5 minutes each at high stringency in 0.1X SSC at 60°C.
If fluorophore-labeled probes were used, proceed to Step 38. In the case of hapten-labeled probes, continue with Step 37.

37. Detect hapten-labeled probes as follows:

i. Incubate the slides in blocking solution (see Step 17.i) for 15 minutes.

ii. Dilute the primary and secondary antibodies (and/or conjugated avidin) in blocking solution.

iii. Incubate the sections with antibodies (and/or conjugated avidin) under coverslips in dark humid containers for 1 hour at 37°C.

iv. After each incubation with antibody (and/or conjugated avidin), wash three times for 5 minutes each with SSC/Tween warmed to 37°C.
Adding saponin and Triton X-100 at a final concentration of 0.1% to all solutions used during the detection of hapten-labeled probes can lead to better penetration of reagents in thick sections, and can decrease background.

38. Incubate slides in 0.05 µg/ml DAPI in SSC/Tween (see Step 18) for 10 minutes. Rinse briefly in 2X SSC.
Alternatively, nuclei can be counterstained with 1 µM TO-PRO-3 for 5 minutes (far-red fluorescence) or with 25 µg/ml PI for 15 minutes (red fluorescence), both in SSC/Tween. Note that PI also stains RNA.

39. Place a drop of Vectashield on top of the section and cover with a coverslip. Avoid making air bubbles. Gently remove excess Vectashield with tissue and seal with nail polish.

40. When the nail polish has dried, examine the preparation under the microscope.
The sections become fragile after FISH. Avoid applying strong pressure to the coverslip.

TROUBLESHOOTING

Problem: There is loss of tissue.

[Step 20]

Solution: Decrease the concentration of and/or length of incubation with protease (see Unmasking DNA).

Problem: The hybridization signal is weak or absent.

[Step 20]

Solution: Try the following:

1. Increase the length of the deparaffinization in xylene to 1 hour (in Step 1).

2. Increase the concentration and/or length of incubation with protease (see Unmasking DNA).

3. Increase the probe concentration used in Step 11.

4. Check the temperature of the heating block used for denaturation (see Step 13 or 14.i).

Problem: There is poor preservation of nuclear morphology.

[Step 20]

Solution: Try the following:

1. Decrease the temperature and/or length of incubation with 1 M sodium isothiocyanate in Step 3.

2. Decrease the concentration and/or length of incubation with protease (see Unmasking DNA).

Problem: The hybridization signal is weak or absent.

[Step 40]

Solutions: Try the following:

1. Increase the number and/or duration of heating pulses during DNA unmasking (see note to Step 26.iv).

2. Increase the length of the permeabilization with acetone in Step 28.

3. Increase the probe concentration used in Step 31.ii.

4. Check the temperature of the heating block used for denaturation in Step 34.

Problem: There is poor preservation of nuclear morphology.

[Step 40]

Solutions: Try the following:

1. Shorten the heating pulses during the DNA unmasking step (see note to Step 26.iv).

2. Make sure that the sodium citrate solution does not boil during Step 26.

Discussion

FISH on histological sections involves balancing two conflicting requirements: ensuring accessibility of probes and antibodies to target DNA, and preserving nuclear morphology. Accessibility of probes is generally not a significant problem, except for larger antibody molecules. To deal with this issue, an "unmasking" step using protease or heat (the latter is easier to control) is included to partially free DNA from cross-linking proteins. This step is crucial to the success of the experiment. It depends on the tissue being analyzed as well as the fixative and embedding medium used during sample processing. Paraffin embedding causes notable deformation of nuclei but is still very commonly used. It is difficult to obtain high-quality FISH signals on paraffin sections of adult human tissues, especially in the case of large diffuse signals typical of chromosome paint probes. Good results are consistently obtained using only fluorophore-labeled probes against repetitive sequences (e.g., alpha-satellites for pericentromeric regions of human chromosomes, Fig. 3 ). Decent signals with hapten-labeled probes can be obtained using the proteinase K pretreatment method on mouse embryonic tissues, which are much less fibrous and compact than adult tissues. Vibratome and cryosections afford good preservation of the nuclear morphology and, therefore, are preferable for nuclear architecture studies (Fig. 4 and Fig. 5 ). Vibratome sections are more permeable than paraffin sections, and cryosections are superior to both.

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Figure 3. FISH on sections of paraffin-embedded tissues (A) Nuclei of human skeletal muscle myotubes counterstained with TO-PRO-3 (red) after hybridization with a probe against human centromeric sequences (labeled with FITC-dUTP; green). Projection of a confocal image stack (~10 µm). (B) Nuclei of human smooth muscle cells counterstained with DAPI (blue) after hybridization with probe for chromosome-specific alphoid DNA and directly labeled with different fluorochromes. Projection of 42 optical sections of a confocal image stack (~12.5 µm). (C) Nuclei of mouse embryonic mesenchymal cells after hybridization with hapten-labeled BAC clones comprising the HoxD gene cluster (red) or HoxC gene cluster (green). Sections were counterstained with DAPI (blue). (Arrows) The two pairs of BAC signals corresponding to Hox genes inside a single ovoid-shaped nucleus. Note that one of the HoxC signals is a doublet (upper right), indicative of DNA replication. Projection of confocal image stacks (~6 µm).

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Figure 4. FISH on vibratome sections (A) Nuclei of ganglion cells of mouse retina counterstained with TO-PRO-3 (blue) after hybridization with a pool of differentially labeled BAC clones comprising either transcriptionally active or inactive genes. BAC DNA was labeled with biotin-dUTP (green) or digoxigenin-dUTP (red). Projection of part of a confocal image stack (~5 µm). (B) Nuclei of bipolar cells of mouse retina counterstained with TO-PRO-3 (blue) after lamin B immunostaining (green) and FISH with a mouse major satellite repeat probe directly labeled with Cy3-dUTP (red). Projection of part of a confocal image stack (~1 µm).

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Figure 5. FISH on cryosections. (A,B) Nuclei of ganglion (A) and bipolar (B) cells of mouse retina counterstained with TO-PRO-3 (blue) after hybridization with a mouse minor satellite probe that recognizes centromeres of mouse chromosomes (directly labeled with Cy3-dUTP, red) and with a probe recognizing telomeres (labeled with biotin-dUTP, green). Projection of a confocal image stack (~4 µm). (C) Nuclei of mouse embryonic neurons after hybridization with a paint probe for mouse chromosome 14 (red) and BAC DNA (green) from the same chromosome. Nucleus was counterstained with DAPI (blue). Projection of part of a confocal image stack (~3 µm). (D) Nuclei of chicken embryonic neurons after hybridization with a paint probe for chicken chromosome 1 (red) and BAC DNA (green) from the same chromosome. Nucleus was counterstained with DAPI (blue). Projection of part of a confocal image stack (~2 µm).

FISH on tissue sections, especially frozen ones, is simple when compared with FISH on cultured cells. When heat is used to "unmask" the target DNA, the technique can be combined with immunostaining to simultaneously locate specific nucleic acids and proteins in the nucleus (e.g., see Fig. 4B). Paraffin-embedded sections are usually 5-10 µm thick, and thus most nuclei on the sections will have to be discarded during analysis because they have been cut one way or another. With the use of thick cryosections (20 µm), a relatively high proportion of nuclei are intact in the middle of the section, thereby facilitating the acquisition of data at the microscope. However, thick sections can also lead to a signal gradient from top to bottom due to poor antibody penetration. To remedy this problem, nonionic detergents can be added to the solutions used in the detection of hapten-labeled probes. Image analysis is another important issue. Some tissues, particularly at embryonic stages, have very tightly packed nuclei. Certain image analysis algorithms, e.g., to measure radial distribution of signals, may require that individual nuclei be isolated from the image stack. This "segmentation" can be accomplished using the Amira software (Mercury Computing Systems), as shown in Figure 6 .

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Figure 6. Segmentation of individual nuclei in image stacks of tissue sections. (A) Single optical section of a confocal image stack of mouse embryonic brain showing densely packed neuronal cells. (B) In this tissue, counterstained nuclei cannot be segmented by straightforward intensity thresholding. (C-F) Individual nuclei can be outlined using the Amira software (v4.0, Mercury Computing Systems). The contour of the nucleus must be outlined by the user on a few optical sections in xy (C), yz (D), and xz (E) planes. Based on this input, the program automatically outlines the surface of the nucleus. These data can be used for 3D rendering (F) or submitted to other programs for quantitative evaluations.

Acknowledgments

The protocols were developed as part of our ongoing studies supported by grants from the Deutsche Forschungsgemeinschaft (Cr 59/20-1-3 and Mu 1850/2-1) and the Bundesministerium für Bildung und Forschung NGFN II- EP (0313377A). C.L. was supported by the Instituts de recherche en santé du Canada and the Humboldt Stiftung.

References

Cremer, M., Müller, S., Köhler, D., Brero, A., and Solovei, I. 2007. Cell preparation and multicolor FISH in 3D preserved cultured mammalian cells. CSH Protocols doi:10.1101/pdb.prot4723.

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., Walter, J., Cremer, M., Habermann, F., Schermelleh, L., and Cremer, T. Beatty, B, ed. 2002. FISH on three-dimensionally preserved nuclei. In FISH: A practical approach, pp. 119–157. Oxford University Press, Oxford, UK.

Walter, J., Joffe, B., Bolzer, A., Albiez, H., Benedetti, P.A., Müller, 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: 367–378.[Medline]