Preparation of Cells from Formalin-Fixed, Paraffin-Embedded Tissue for Use in Fluorescence In Situ Hybridization (FISH) Experiments
UNIT 8.8
Stanislawa Weremowicz1 1
CAMD-Cytogenetics, Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts
Numerical and structural chromosome abnormalities can be accurately detected in cells from archived tissues using fluorescence in situ hybridization (FISH). This unit describes two common approaches to performing FISH in formalinfixed, paraffin-embedded tissue. The first approach utilizes 4 to 6 μm tissue sections in cases for which preserving tissue morphology is necessary, and the second involves extraction of intact nuclei from 50-μm tissue sections. To interpret FISH results using 4 to 6 μm sections, an adequate number of nuclei must be evaluated to perform statistical analysis. Evaluation of 30 to 50 nuclei C 2015 from the single-cell suspension generally gives an interpretable result. by John Wiley & Sons, Inc. Keywords: interphase cytogenetics r FISH r archived tissue
How to cite this article: Weremowicz, S. 2015. Preparation of Cells from Formalin-Fixed, Paraffin-Embedded Tissue for Use in Fluorescence In Situ Hybridization (FISH) Experiments. Curr. Protoc. Hum. Genet. 84:8.8.1-8.8.10. doi: 10.1002/0471142905.hg0808s84
INTRODUCTION Fluorescence in situ hybridization (FISH; UNIT 4.3; Knoll and Lichter, 2005) is a molecularbased technique that allows rapid and accurate detection of specific numerical or structural abnormalities in a variety of samples. Early FISH applications, aimed at detection of numerical abnormalities in mitotic or interphase cells and structural abnormalities in metaphase chromosome spreads, were followed by strategies for identification of structural chromosome abnormalities in interphase cells (Tkachuk et al., 1990). In most cases FISH is performed on cells obtained from cell cultures, suspensions of fresh subsequently fixed cells, or frozen tissue. However, occasionally fresh or cultured cells are unavailable; therefore successful application of FISH to cells from routinely processed formalin-fixed, paraffin-embedded tissue (Emmerich et al., 1989; Walt et al., 1989; Hopman et al., 1991) helps to circumvent this problem. Subsequently, paraffin blocks became an alternative source of cells for interphase cytogenetics. Paraffin blocks, routinely stored up to 20 years, are an excellent source of cells with potentially useful clinical information. There are two common approaches to performing FISH in formalin-fixed, paraffinembedded tissue. The first utilizes unstained 4 to 6 μm tissue sections and is particularly helpful in cases for which preserving tissue morphology is either important or necessary (Basic Protocol 1). The second approach involves extraction of intact nuclei from Clinical Cytogenetics Current Protocols in Human Genetics 8.8.1-8.8.10, January 2015 Published online January 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142905.hg0808s84 C 2015 John Wiley & Sons, Inc. Copyright
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50-μm tissue sections in cases for which information about the spatial relationship of cells is unnecessary (Basic Protocol 2). Both of these techniques work well on tissues fixed in formalin or paraformaldehyde. Other fixatives may be used (e.g., alcohol), yet others are considered incompatible with successful FISH (see Critical Parameters for further discussion). BASIC PROTOCOL 1
PREPARATION OF PARAFFIN SECTIONS FOR FISH This protocol can be applied in cases when preservation of tissue architecture is necessary. Paraffin sections (4 to 6 μm) are placed on silanized or positively charged glass microscope slides, deparaffinized, treated with a proteolytic enzyme, then dehydrated and hybridized with a selected DNA probe. The hybridized probe is detected by a fluorescent method then viewed and analyzed with fluorescence microscopy (UNIT 4.3; Knoll and Lichter, 2005).
Materials Paraffin-embedded tissue of interest Silanized glass (precleaned), or positively charged, microscope slides (see recipe) Xylene or Hemo-De (Scientific Safety Solvents) Ethanol Sodium bisulfite (Sigma-Aldrich) 2× sodium chloride/sodium citrate (SSC), pH 7.0 (see APPENDIX 2D for 20× SSC) 25 mg/ml proteinase K (Sigma-Aldrich; store at –20°C) or pepsin (Paraffin Pretreatment Kit 1, Abbott Molecular; store at –20°C) 0.6 μg/ml propidium iodide/Antifade (MP Biomedicals) DNA probes (Abbott Molecular, MP Biomedicals, Cytocell Ltd.) Hybridization buffer (MP Biomedicals; LSI or CEP, Abbott Molecular) Rubber cement 0.3% (v/v) Tween 20 in 0.4× SSC (pH 7.0) 0.1% (v/v) Tween 20 in 2× SSC (pH 7.0) Phosphate-buffered detergent (PBD; MP Biomedicals), room temperature 125 mg/ml 4 ,6-diamidino-2-phenylindole (DAPI) II/Antifade solution (Abbott Molecular) Filter set: Green/Red dual band (e.g., cat. no. 59010, Chroma Technology Corp.; see also UNIT 4.4; McNamara et al., 2005) Aqua/Green/Red triple band (e.g., cat. no. 69008, Chroma Technology Corp.; see also UNIT 4.4; McNamara et al., 2005) Microtome 65°C and 95°C ovens (or a slide warmer; e.g., HYBrite, Abbott Molecular, Leica Biosystems) 50-ml glass Coplin jars Epifluorescence microscope equipped with 100-W mercury lamp (UNIT 4.4; McNamara et al., 2005) 20× objective and 40× or 100× oil immersion fluorescence objectives 22 × 22-mm glass coverslips Humidified hybridization chamber (UNIT 4.3; Knoll and Lichter, 2005) 24 × 50-mm glass no.1 coverslips Preparation of Cells from Fixed Tissue for Use in FISH Experiments
Additional reagents and equipment for paraffin embedding and preparation of tissue sections (Zeller, 1989), FISH (UNIT 4.3; Knoll and Lichter, 2005), and fluorescence microscopy (UNIT 4.4; McNamara et al., 2005)
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Prepare slides 1. Using a microtome, cut 4- to 6-μm sections of paraffin-embedded tissue and place on silanized glass microscope slides by floating the sections in water. Optimal thickness of tissue sections will depend on tissue cellularity, amount of connective tissue and nuclear diameter (5- to 15-μm is an average nuclear diameter); 4- to 6-μm sections are commonly used. Sections thicker than 7 μm may require confocal microscopy for evaluation of hybridization signals.
2. Air dry slides and bake 2 hr or overnight at 65°C. This step is necessary to ensure that the tissue sections adhere to the slides in the subsequent steps.
Deparaffinize the sample 3. Deparaffinize tissue section in 50 ml xylene (use glass Coplin jars) or in Hemo-De for 10 min, followed by two washes in 100% ethanol for 5 min each, while agitating occasionally. Air dry slides. Treat sample with proteolytic enzyme 4. Treat with sodium bisulfite solutions (10%, 20%, or 30% [w/v] in 2× SSC, pH 7.0) for 10 to 30 min at 45°C. Rinse briefly in 2× SSC, pH 7.0. Theoretically, sodium bisulfite or sodium isothiocyanate facilitates access of proteolytic enzymes to the tissue. The concentration of sodium bisulfite solution and the treatment time must be determined empirically. Start with 20% solution for 15 min and adjust for a particular tissue type. Tissues such as spleen, kidney, and heart may require a higher concentration and a longer treatment time compared to skin, gut or lung. IMPORTANT NOTE: Working solution of sodium bisulfite must be used the same day as it is prepared.
5. Add 400 μl of 25 mg/ml proteinase K to 40 ml of 2× SSC, pH 7.0. Incubate slides in this proteinase K working solution 10 to 30 min at 45°C. IMPORTANT NOTE: Working solution of proteinase K must be used the same day as it is prepared. The proteinase K concentration and treatment time must be determined empirically. Start with 10 min incubation time. Alternatively, a commercially available pretreatment kit (i.e., Abbott Molecular) can be used according to the manufacturer’s instructions.
6. Rinse slides in three changes of 2× SSC, pH 7.0 for 1 min each at room temperature.
Dehydrate the sample 7. Dehydrate slides in 70%, 80%, 90%, and 100% ethanol by immersing for 2 min each at room temperature. Air dry slides 8. Stain slides with propidium iodide/Antifade (apply 10 μl and cover with a 22 × 22mm glass coverslip) and evaluate using fluorescence microscopy (UNIT 4.4; McNamara et al., 2005) to determine the degree of digestion. Over-digestion will cause loss of nuclear borders, may give a “ghostly” appearance of nuclei, and ultimately lead to a loss of material altogether. Persistent green autofluorescence and poor propidium iodide staining indicate that the tissue is underdigested. In this case, slides should be transferred to 2× SSC, pH 7.0, for several minutes before repeating steps 4 and 5.
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22 X 22–mm glass coverslip
sample nuclei
rubber cement
glass slide
Figure 8.8.1 Placement of sample nuclei on glass slide to allow two separate hybridizations, with two probes or two sets of probes, to be performed simultaneously.
Prepare probe 9. Prepare DNA probes as described in UNIT 4.3 (Knoll and Lichter, 2005). Preparation of DNA probes including labeling, use of Cot1 DNA, along with concentration guidelines and methods for detection of the hybridized probe are described in UNIT 4.3 (Knoll and Lichter, 2005). For commercial probes, follow manufacturer’s instruction. In general, the concentration of probe depends on the size and complexity of the target sequence. Five to twenty ng of probe/μl of hybridization buffer usually works well (e.g., 5 to 10 ng/μl for whole chromosome paint or cosmid probes, 15 to 20 ng/μl for P1, BAC, YAC probes). Ten μl of hybridization mixture (i.e., hybridization buffer + probe) is sufficient to cover the area under 22 × 22-mm coverslip. IMPORTANT NOTE: Store probes at –20°C and protect directly labeled probes (e.g., Spectrum Orange, Spectrum Green) from light. Prolonged storage of the hybridization mixture at room temperature prior to application to the sample should be avoided.
Hybridize probe to the sample 10. Pipet 10 μl of hybridization mixture onto target area, cover with glass coverslip, and seal with rubber cement (Fig. 8.8.1). If using two different probes on the same slide, apply one probe, coverslip (usually a 22 × 22-mm coverslip works best), and sealant before applying the second probe to prevent the probes from mixing (see Fig. 8.8.1).
11. Denature probe and target DNA at 95°C for 10 min. Depending on the number of slides, it may be necessary to heat the oven up to 100°C to maintain 95°C. In general, a 10-min denaturation time works well. However, if the nuclei are fragile due to some unknown factor of tissue fixation or storage, denaturation times can be shortened to 5 min or temperatures decreased to as low as 75°C with minimal loss of final hybridization signal intensity. Alternatively, a slide warmer (e.g., HYBrite) can be used for denaturation.
12. Incubate slides in a humidified chamber at 37°C or 42°C overnight to allow hybridization to occur.
Wash off excess probe 13. Remove coverslips and wash slides, in 50-ml glass Coplin jars, as follows: a. Once in 0.4× SSC pH 7.0/0.3% (v/v) Tween 20 at 73°C for 2 min. b. Once in 2× SSC pH 7.0/0.1% (v/v) Tween 20 at room temperature for 1 min. Preparation of Cells from Fixed Tissue for Use in FISH Experiments
This wash procedure usually yields preparation with a low signal-to-noise ratio. If an excess background is observed, repeat wash and more frequent agitation of slides is recommended. For probes that yield a relatively weak hybridization signal, lower wash
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stringency is recommended (e.g., 50% formamide/2× SSC, 2× SSC at 39°C or 42°C, UNIT 4.3; Knoll and Lichter, 2005).
14. Transfer slides to Coplin jar containing 1× PBD at room temperature. At this point the slides can be stored in 1× PBD over night at 4°C if necessary. The manual procedure described above can be automated by using the ThermoBrite Elite FISH Auto Slide Prep (Leica Biosystems).
Detect hybridized probe Detection and amplification of hybridization signals of biotin- or digoxigenin-labeled probes is described in detail in UNIT 4.3 (Knoll and Lichter, 2005). No detection is required for directly labeled probes. 15. Counterstain with DAPI II/Antifade solution (10 μl) and cover with a 24 × 50-mm no.1 glass coverslip. 16. Examine slides with a fluorescence microscope (UNIT 4.4; McNamara et al., 2005) using the appropriate filter set. Record the hybridization pattern or number of hybridization signals per nucleus.
PREPARATION OF SINGLE-CELL SUSPENSION FROM FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUE FOR FISH
BASIC PROTOCOL 2
This protocol requires 50-μm tissue sections cut from selected areas of paraffin blocks. The tissue is deparaffinized, rehydrated, and disaggregated using proteolytic enzymes. The protocol is based on a method described by Kuchinka et al. (1995), with modifications as described below.
Materials Paraffin-embedded tissue of interest Silanized glass (precleaned), or positively charged, microscope slides (see recipe) Xylene Ethanol Hanks’ balanced salt solution (HBSS; APPENDIX 2D) 1 mg/ml collagenase XI solution (Sigma-Aldrich) 0.05% trypsin/EDTA (Invitrogen) Sterile H2 O Microtome 15-ml glass conical centrifuge tubes Centrifuge (e.g., Marathon 3000, Fisher Scientific) Vortex 50°C oven (or slide warmer; e.g., HYBrite, Abbott Molecular, Leica Biosystems) Additional reagents and equipment for probe hybridization (see Basic Protocol 1) Deparaffinize and rehydrate the sample 1. Using a microtome, cut two to five 50-μm sections and place in 15-ml glass conical centrifuge tubes. The number of tissue sections needed depends upon both the size and cellularity of the material; for most types of tissue, two 1 × 1-cm sections should yield enough nuclei to perform hybridization with 10 probes.
2. Add 5 ml xylene and let stand 5 min, agitate occasionally, then centrifuge 30 to 60 sec at 287 × g, room temperature. Discard supernatant.
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The number of specimens that can be disaggregated at one time is limited by both the number of tubes that the centrifuge holds and manual constraints later in the procedure. In general, it is cumbersome to handle >10 specimens per run.
3. Repeat step 2 using the following solutions in turn:
5 ml xylene 5 ml 100% ethanol 5 ml 100% ethanol 5 ml 80% ethanol 5 ml 50% ethanol 5 ml sterile H2 O Leave in water overnight at 4°C At this point visual inspection of the tube should show no evidence of white waxy paraffin flecks. If a few fragments of paraffin are observed, they can be removed manually. If paraffin fragments cannot be removed, the tissue should be dehydrated by repeating the wash and centrifuge steps described in steps 2 and 3 above but using the solvents in reverse order (i.e., 50% ethanol, 80% ethanol, 100% ethanol, 100% ethanol, xylene, xylene). Increasing xylene treatment time with occasional agitation is recommended. Repeat step 3 to rehydrate the tissue.
4. Centrifuge for 30 to 60 sec at 287 × g, room temperature. Discard supernatant. 5. Wash 1× in HBSS. Centrifuge for 30 to 60 sec at 287 × g, room temperature, and discard supernatant.
Extract nuclei 6. Add 1 ml collagenase XI solution (1 mg/ml) and incubate at 37°C for 2 hr. Add 9 ml HBSS. Centrifuge for 30 to 60 sec at 287 × g, room temperature, then discard supernatant. 7. Add 5 ml of 0.05% trypsin/EDTA; incubate at 37°C for 1 hr or longer. Centrifuge for 30 to 60 sec at 287 × g, room temperature, and discard supernatant. For previously untested tissue start with 30 min of trypsin treatment and check for disaggregation. A 1-hr incubation usually gives good results.
8. Wash tissue with 5 ml HBSS once. Centrifuge for 30 to 60 sec at 287 × g, room temperature, discard supernatant and then add 1 ml HBSS. Vortex vigorously and allow large pieces of tissue to settle (1 to 2 min). 9. Drop cell suspension on slide using a Pasteur pipet. Single cells should be visible by phase-contrast microscopy. Check cell concentration and dilute if necessary by adding more HBSS.
Prepare slide 10. Place a drop of cell suspension on one or two areas of a glass slide (Fig. 8.8.1), bake for 2 hr or overnight at 50°C. Transfer remaining cell suspension to a microcentrifuge tube and store at 4°C. For longer storage remove supernatant and resuspend pellet in 70% ethanol. Store at −80°C.
Preparation of Cells from Fixed Tissue for Use in FISH Experiments
Dehydrate the sample 11. Dehydrate slides by immersing in 70%, 80%, 90%, and 100% ethanol for 2 min each at room temperature and allow to air dry. The slides are ready to use for hybridization, or they can be stored in a dry chamber for several weeks. If stored, dehydrate the slide again before use.
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Hybridize probe to the sample 12. Hybridize, wash and examine slides as described in Basic Protocol 1, steps 10 to 14. REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2D; for suppliers, see SUPPLIERS APPENDIX.
Silanized glass microscope slides Prepare a 2% (v/v) silanization solution by mixing 2 ml of 3aminopropyltriethoxysilane (Sigma-Aldrich) and 98 ml acetone. Dip slides into silanization solution for 20 sec. Wash in two changes of acetone, 15 sec each, then air dry. Store slides indefinitely at room temperature.
COMMENTARY Background Information Routine cytogenetics based on chromosomal banding techniques has been successful in correlating constitutional or acquired karyotype abnormalities with clinical diagnosis, prognosis, and, in some cases, therapeutic response. However, this requires a source of viable, mitotic cells with good chromosome morphology and usually involves a relatively long turn-around time. The advent of molecular cytogenetics, with development and optimization of fluorescence in situ hybridization (FISH), brought considerable progress especially in cancer cytogenetics, and at present the technique is widely used for both research and clinical studies (see UNIT 4.3; Knoll and Lichter, 2005). DNA segments of interest serve as probes after a fluorescent tag or a reporter molecule is attached. Under proper hybridization conditions the probe recognizes and binds to the homologous sequences in target DNA. After hybridization, the target homologous regions are recognized and evaluated by using fluorescence microscopy. The technology is simple and robust and its significant contribution, especially to cancer cytogenetics, relies on its applicability to interphase cells. Thanks to the Human Genome Project, scores of genomic DNA fragments were fully characterized and mapped to specific chromosomes and chromosome regions. This allowed creation of YAC, BAC, cosmid or plasmid libraries, which serve as an excellent source of specific DNA probes in addition to those previously employed probes comprised of alpha, beta or classical satellite DNAs. Most FISH studies are performed on cells from fresh or frozen tissues. However, it has been well documented that formalin-fixed, paraffin-embedded tissue can provide a viable alternative (Schofield and Fletcher, 1992;
Kuchinka et al., 1995; Di Francesco et al., 2000; Pickering et al., 2001; Gelpi et al., 2003) when fresh tissue is not available. The use of fixed tissues increases the possibility of analyzing large number of archival cases of rare anomalies. FISH analysis of formalin-fixed, paraffinembedded tissue can be performed on unstained histological tissue sections or on disaggregated, intact nuclei. Unstained sections should be used when preservation of tissue morphology is necessary. A limitation of this technique is that it results in many incomplete nuclei, which can lead to a loss of some chromosomal material and subsequently to underestimation of chromosome copy number. Interpretation of results requires statistical analysis of the FISH signals observed in both normal tissue and the clinical specimen and establishing laboratory cut-offs for aneuploidy evaluation (Qian et al., 1996) or target-toreference probe ratio in cases tested for a loss of a target allele (Gelpi et al., 2003). However, this method allows precise histopathological correlation of multiple foci of normal cells, premalignant lesions, and tumor cells within a single specimen, including study of intratumor heterogeneity. The second approach involves extraction of intact nuclei from 50-μm tissue sections. The method eliminates the problem of loss of chromosomal material due to sectioning of nuclei and can be used to evaluate both numerical and structural chromosome abnormalities (Kuchinka et al., 1995; Qian et al., 1996; Pickering et al., 2001). The main disadvantage is loss of the spatial relationships of the cells and possible admixture of normal stromal cells. This problem can be circumvented by using hematoxylin and eosin (H & E) stained sections and careful evaluation of
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selected paraffin blocks to identify the best areas for sampling (e.g., highest percentage of tumor cells, fetal parts of placenta, areas free of necrosis; DiFrancesco et al., 2001; Pickering et al., 2001; Gelpi et al., 2003). FISH analysis of isolated nuclei does not require special training in pathology, and counting/evaluation of fluorescence signals is easier on isolated nuclei than on tissue sections. Finally, large-scale high-throughput adaptation of this technique is feasible.
Critical Parameters and Troubleshooting
Preparation of Cells from Fixed Tissue for Use in FISH Experiments
Tissue fixation Optimally, tissue samples should be fixed in neutral-buffered formalin or paraformaldehyde for 24 to 40 hr. Parameters such as fixation time, block size, and the embedding process all can affect FISH analysis. Prolonged fixation time may impair access of probe to the target DNA due to excessive cross-linking between nuclear proteins and between proteins and DNA. Tissue quality is important; therefore delay in fixation time, leading to decay and autolysis of the tissue, may cause weak or absent hybridization signals. Preparation of paraffin sections from specimens containing bone or calcified tissue areas (e.g., dystrophic calcification, metastatic calcification) requires thorough fixation to protect cells and fibrous elements of the tissue from damage caused by decalcifying agents (e.g., acids, chelating agents) during the subsequent decalcifying process. For fixation, buffered formalin is preferable; however, in some cases where preservation of a specific tissue (e.g., bone marrow) is important, alternative fixatives such as B5 or Bouin’s are used. Commercial suppliers of DNA probes consider some fixatives including B5, Zenker’s, Zamboni’s, Bouin’s, and mercuric chloride as incompatible with standard FISH protocols. Prolonged treatment with decalcifying agents can cause DNA degradation; therefore, FISH analysis on routinely prepared paraffin sections from specimens containing bone or calcified tissue areas might be unsuccessful. If FISH testing is needed on tissue sections from such specimens, prolonged treatment with decalcifying agents and/or application of fixatives incompatible with FISH must be avoided, and the protocol(s) will require modifications. For example, a limited acid decalcification with 5% formic acid (Brown et al., 2002), ultrasonic decalcification (Reineke et al., 2006), or alternatively, closely monitored decalcification us-
ing EDTA (Zustin et al., 2009) protocols can be applied, and a method described by Schurter et al. (2002) can be used when FISH is performed following use of one of the FISH-incompatible fixatives. Tissue pretreatment/digestion Removal of proteins via pretreatment with sodium bisulfite solution and digestion with proteolytic enzymes (e.g., proteinase K, pepsin) or other commercial pretreatment solutions (from, e.g., Vysis, Inc., MP Biochemicals) reduces autofluorescence, enhances accessibility of target DNA, and may lead to improvement of hybridization signal intensity. Concentration of proteinase K or pepsin is critical because for both, insufficient or excess digestion will affect hybridization. Underdigestion leads to autofluorescence; overdigestion will compromise nuclear morphology and/or cause loss of the tissue. Incubation time can vary depending on tissue type and length of fixation time. Therefore, specific treatment times must be determined empirically from case to case or tissue to tissue. For in vitro diagnostic commercial products, the manufacturer’s protocols and recommendations should be followed. During preparation of the single-cell suspensions (Basic Protocol 2) potential problems include insufficient numbers or poor morphology of nuclei. Insufficient numbers of nuclei can be a result of a hypocellular tissue sample or incomplete tissue digestion. The specimen should be re-evaluated and additional sections from the same or different block should be obtained to repeat the experiment. If hypocellularity is not a problem, then digestion time should be increased. Poor morphology of nuclei or excessive nuclear/cellular debris can be a result of poor sample quality (necrosis) or over-digestion. Re-evaluation of the tissue block and/or decreased digestion time should be considered. In situ hybridization For problems related to in situ hybridization (background, cross-hybridization, etc.) see UNIT 4.3 (Knoll and Lichter, 2005). Efficiency of hybridization and hybridization signals can be affected by the quality of tissue sample, fixation time, and type of fixative. To some degree, such problems can be solved by modifying the standard protocol (Hyytinen et al., 1994; Schurter et al., 2002); however, in some cases they may remain intractable. Increasing the denaturation time can be considered.
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Anticipated Results Hybridization efficiency of over 90% can be achieved for nuclei prepared from tissue samples collected at the time of surgery and processed appropriately. For samples collected at autopsy, hybridization results are more variable, and in some cases the nuclei may fail to hybridize. To interpret FISH results using Basic Protocol 1, an adequate number of nuclei must be evaluated before performing appropriate statistical analysis such as a Student’s t-test and/or chi-square test (Weremowicz et al., 1994; Qian et al., 1996; Le Maitre et al., 2001) to compare test results to control probes, or normal controls, and to establish laboratory cut-offs. Evaluation of 30 to 50 nuclei from single-cell suspensions (Basic Protocol 2) generally gives an interpretable result (S. Weremowicz, unpub. observ.).
Time Considerations Both protocols are labor intensive and time consuming. FISH results on tissue sections (Basic Protocol 1) can be available in 72 hr and on disaggregated cells (Basic Protocol 2) in 96 hr. However, the opportunity to evaluate archival material and to acquire valuable clinical information, otherwise not obtainable, far outweighs the labor requirement. To decrease turn-around time, the FISH procedure on tissue sections might be modified by applying a microwave irradiation method (Kitayma et al., 2000; Ridderstrale et al., 2005).
Literature Cited Brown, R.S.D, Edwards, J., Bartlett, J.W., Jones, C., and Dogan, A. 2002. Routine acid decalcification of bone marrow samples can preserve DNA for FISH and CGH studies in metastatic prostate cancer. J. Histochem. Cytochem. 50:113-115. Di Francesco, L.M., Murthy, S.K., Luider, J., and Demetrick, D.J. 2000. Laser capture microdissection-guided fluorescence in situ hybridization and flow cytometric cell cycle analysis of purified nuclei from paraffin sections. Mod. Pathol. 13:705-711. Emmerich, P., Jauch, A., Hofmann, M.-C., Cremer, T., and Walt, H. 1989. Interphase cytogenetics in paraffin embedded sections from testicular germ cell tumor xenografts and in corresponding cultured cells. Lab. Invest. 61:235-242. Gelpi, E., Ambros, I.M., Birner, P., Leugmayr, A., Drlicek, M., Fischer, I., Kleinert, R., Maier, H., Huemer, M., Gatterbauer, B., Anton, J., Rossler, K., Budka, H., Ambros, P.F., and Hainfellner, J.A. 2003. Fluorescent in situ hybridization on isolated tumor cell nuclei: A sensitive method for 1p and 19q deletion analysis in paraffinembedded oligodendrial tumor specimens. Mod. Pathol. 16:708-715.
Hopman, A.H.N., van Hooren, E., van de Kaa, C.A., Vooijs, P.G.P., and Ramaekers, F.C.S. 1991. Detection of numerical chromosome aberrations using in situ hybridization in paraffin sections of routinely processed bladder cancers. Mod. Pathol. 4:503-513. Hyytinen, E., Visakorpi, T., Kallioniemi, A., Kallioniemi, O.-P., and Isola, J.J. 1994. Improved technique for analysis of formalin-fixed, paraffin-embedded tumors by fluorescence in situ hybridization. Cytometry 16:93-99. Kitayama, Y., Igarashi, H., and Sugimura, H. 2000. Initial intermittent microwave irradiation for fluorescent in situ hybridization analysis in paraffin-embedded tissue sections of gastrointestinal neoplasia. Lab. Invest. 80:779781. Knoll, J.H.M. and Lichter, P. 2005. In situ hybridization to metaphase chromosomes and interphase nuclei. Curr. Protoc. Hum. Genet. 45:4.3.1-4.3.31. Kuchinka, B.D., Kalousek, D.K., Lomax, B.L., Harrison, K.J., and Barrett, I.J. 1995. Interphase cytogenetic analysis of single cell suspensions prepared from previously formalin-fixed and paraffin-embedded tissues. Mod. Pathol. 8:183186. Le Maitre, C.L., Byers, R.J., Liu Yin, J.A., Hoyland, J.A., and Freemont, A.J. 2001. Dual colour FISH in paraffin bone trepines for identification of numerical and structural chromosome abnormalities in acute myeloid leukemia and myelodysplasia. J. Clin. Pathol. 54:730-733. McNamara, G., Difilippantonio, M.J. and Ried, T. 2005. Microscopy and image analysis. Curr. Protoc. Hum. Genet. 46:4.4.1-4.4.34. Pickering, D.L., Nelson, M., Chan, W.C., Huang, J.Z., Dave, B.J., and Sanger, W.G. 2001. Paraffin tissue core sectioning: An improved technique for whole nuclei extraction and interphase FISH. J. Assoc. Gen. Tech. 27:38-40. Qian, J., Bostwik, D.G., Takahashi, S., Borell, T.J., Brown, J.A., Lieber, M.M., and Jenkins, R.B. 1996. Comparison of fluorescence in situ hybridization analysis of isolated nuclei and routine histological sections from paraffinembedded prostatic adenocarcinoma specimens. Am. J. Pathol. 49:1193-1199. Reineke, T., Jenni, B., Abdou, M.-T., Frigerio, S., Zubler, P., Moch, H., and Tinguely, M. 2006. Ultrasonic decalcification offers new perspectives for rapid FISH, DNA, and RT-PCR analysis in bone marrow trephines. Am. J. Surg. Pathol. 30:892-896. Ridderstrale, K.K., Grushko, T.A., Kim, H.-J., and Olopade, O.I. 2005. Single-day FISH procedure for paraffin embedded tissue sections using a microwave oven. BioTechniques 39:316320. Schofield, D.E. and Fletcher, J.A. 1992. Trisomy 12 in pediatric granulosa-stromal cell tumors: Demonstration by a modified method of fluorescence in-situ hybridization on paraffinembedded material. Am. J. Pathol. 141:12651269.
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Schurter, M.J., LeBrun, D.P., and Harrison, K.J. 2002. Improved technique for fluorescence in situ hybridisation analysis of isolated nuclei from archival, B5 or formalin fixed, paraffin wax embedded tissue. J. Clin. Pathol. Mol. Pathol. 55:121-124. Tkachuk, D.C., Westbrook, C., Andreef, M., Donlon, T.A., Clearly, M.L., Suryanaryan, K., Homge, M., Redner, A., Gray, J., and Pinkel, D. 1990. Detection of bcr-abl fusion in chronic myelogenous leukemia by in situ hybridization. Science 250:559-562. Walt, H., Emmerich, P., Cremer, T., Hofmann, M.-C., and Bannwart, F. 1989. Supernumerary chromosome 1 in interphase nuclei of atypical germ cells in paraffin-embedded hu-
man seminiferous tubules. Lab. Invest. 61:527531. Weremowicz, S., Kozakewich, H.P., Haber, D., Park, S., Morton, C.C., and Fletcher, J.A. 1994. Identification of genetically aberrant cell lineages in Wilms’ tumors. Genes Chromosomes Cancer 10:40-48. Zeller, R. 1989. Fixation, embedding, and sectioning of tissues, embryos, and single cells. Curr. Protoc. Mol. Biol. 7:14.1.1-14.1.8. Zustin, J., Baddin, K., Tsourlakis, M.C., Burandt, E., Mirlacher, M., Jaenicke, F., Izbicki, J., Reuther, W., Rueger, J.M., Bokemeyer, C., Simon, R., and Sauter, G. 2009. HER2/neu analysis in breast cancer bone metastases. J. Clin. Pathol. 62:542-546.
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UNIT 8.8
Stanislawa Weremowicz1 1
CAMD-Cytogenetics, Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts
Numerical and structural chromosome abnormalities can be accurately detected in cells from archived tissues using fluorescence in situ hybridization (FISH). This unit describes two common approaches to performing FISH in formalinfixed, paraffin-embedded tissue. The first approach utilizes 4 to 6 μm tissue sections in cases for which preserving tissue morphology is necessary, and the second involves extraction of intact nuclei from 50-μm tissue sections. To interpret FISH results using 4 to 6 μm sections, an adequate number of nuclei must be evaluated to perform statistical analysis. Evaluation of 30 to 50 nuclei C 2015 from the single-cell suspension generally gives an interpretable result. by John Wiley & Sons, Inc. Keywords: interphase cytogenetics r FISH r archived tissue
How to cite this article: Weremowicz, S. 2015. Preparation of Cells from Formalin-Fixed, Paraffin-Embedded Tissue for Use in Fluorescence In Situ Hybridization (FISH) Experiments. Curr. Protoc. Hum. Genet. 84:8.8.1-8.8.10. doi: 10.1002/0471142905.hg0808s84
INTRODUCTION Fluorescence in situ hybridization (FISH; UNIT 4.3; Knoll and Lichter, 2005) is a molecularbased technique that allows rapid and accurate detection of specific numerical or structural abnormalities in a variety of samples. Early FISH applications, aimed at detection of numerical abnormalities in mitotic or interphase cells and structural abnormalities in metaphase chromosome spreads, were followed by strategies for identification of structural chromosome abnormalities in interphase cells (Tkachuk et al., 1990). In most cases FISH is performed on cells obtained from cell cultures, suspensions of fresh subsequently fixed cells, or frozen tissue. However, occasionally fresh or cultured cells are unavailable; therefore successful application of FISH to cells from routinely processed formalin-fixed, paraffin-embedded tissue (Emmerich et al., 1989; Walt et al., 1989; Hopman et al., 1991) helps to circumvent this problem. Subsequently, paraffin blocks became an alternative source of cells for interphase cytogenetics. Paraffin blocks, routinely stored up to 20 years, are an excellent source of cells with potentially useful clinical information. There are two common approaches to performing FISH in formalin-fixed, paraffinembedded tissue. The first utilizes unstained 4 to 6 μm tissue sections and is particularly helpful in cases for which preserving tissue morphology is either important or necessary (Basic Protocol 1). The second approach involves extraction of intact nuclei from Clinical Cytogenetics Current Protocols in Human Genetics 8.8.1-8.8.10, January 2015 Published online January 2015 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142905.hg0808s84 C 2015 John Wiley & Sons, Inc. Copyright
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50-μm tissue sections in cases for which information about the spatial relationship of cells is unnecessary (Basic Protocol 2). Both of these techniques work well on tissues fixed in formalin or paraformaldehyde. Other fixatives may be used (e.g., alcohol), yet others are considered incompatible with successful FISH (see Critical Parameters for further discussion). BASIC PROTOCOL 1
PREPARATION OF PARAFFIN SECTIONS FOR FISH This protocol can be applied in cases when preservation of tissue architecture is necessary. Paraffin sections (4 to 6 μm) are placed on silanized or positively charged glass microscope slides, deparaffinized, treated with a proteolytic enzyme, then dehydrated and hybridized with a selected DNA probe. The hybridized probe is detected by a fluorescent method then viewed and analyzed with fluorescence microscopy (UNIT 4.3; Knoll and Lichter, 2005).
Materials Paraffin-embedded tissue of interest Silanized glass (precleaned), or positively charged, microscope slides (see recipe) Xylene or Hemo-De (Scientific Safety Solvents) Ethanol Sodium bisulfite (Sigma-Aldrich) 2× sodium chloride/sodium citrate (SSC), pH 7.0 (see APPENDIX 2D for 20× SSC) 25 mg/ml proteinase K (Sigma-Aldrich; store at –20°C) or pepsin (Paraffin Pretreatment Kit 1, Abbott Molecular; store at –20°C) 0.6 μg/ml propidium iodide/Antifade (MP Biomedicals) DNA probes (Abbott Molecular, MP Biomedicals, Cytocell Ltd.) Hybridization buffer (MP Biomedicals; LSI or CEP, Abbott Molecular) Rubber cement 0.3% (v/v) Tween 20 in 0.4× SSC (pH 7.0) 0.1% (v/v) Tween 20 in 2× SSC (pH 7.0) Phosphate-buffered detergent (PBD; MP Biomedicals), room temperature 125 mg/ml 4 ,6-diamidino-2-phenylindole (DAPI) II/Antifade solution (Abbott Molecular) Filter set: Green/Red dual band (e.g., cat. no. 59010, Chroma Technology Corp.; see also UNIT 4.4; McNamara et al., 2005) Aqua/Green/Red triple band (e.g., cat. no. 69008, Chroma Technology Corp.; see also UNIT 4.4; McNamara et al., 2005) Microtome 65°C and 95°C ovens (or a slide warmer; e.g., HYBrite, Abbott Molecular, Leica Biosystems) 50-ml glass Coplin jars Epifluorescence microscope equipped with 100-W mercury lamp (UNIT 4.4; McNamara et al., 2005) 20× objective and 40× or 100× oil immersion fluorescence objectives 22 × 22-mm glass coverslips Humidified hybridization chamber (UNIT 4.3; Knoll and Lichter, 2005) 24 × 50-mm glass no.1 coverslips Preparation of Cells from Fixed Tissue for Use in FISH Experiments
Additional reagents and equipment for paraffin embedding and preparation of tissue sections (Zeller, 1989), FISH (UNIT 4.3; Knoll and Lichter, 2005), and fluorescence microscopy (UNIT 4.4; McNamara et al., 2005)
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Prepare slides 1. Using a microtome, cut 4- to 6-μm sections of paraffin-embedded tissue and place on silanized glass microscope slides by floating the sections in water. Optimal thickness of tissue sections will depend on tissue cellularity, amount of connective tissue and nuclear diameter (5- to 15-μm is an average nuclear diameter); 4- to 6-μm sections are commonly used. Sections thicker than 7 μm may require confocal microscopy for evaluation of hybridization signals.
2. Air dry slides and bake 2 hr or overnight at 65°C. This step is necessary to ensure that the tissue sections adhere to the slides in the subsequent steps.
Deparaffinize the sample 3. Deparaffinize tissue section in 50 ml xylene (use glass Coplin jars) or in Hemo-De for 10 min, followed by two washes in 100% ethanol for 5 min each, while agitating occasionally. Air dry slides. Treat sample with proteolytic enzyme 4. Treat with sodium bisulfite solutions (10%, 20%, or 30% [w/v] in 2× SSC, pH 7.0) for 10 to 30 min at 45°C. Rinse briefly in 2× SSC, pH 7.0. Theoretically, sodium bisulfite or sodium isothiocyanate facilitates access of proteolytic enzymes to the tissue. The concentration of sodium bisulfite solution and the treatment time must be determined empirically. Start with 20% solution for 15 min and adjust for a particular tissue type. Tissues such as spleen, kidney, and heart may require a higher concentration and a longer treatment time compared to skin, gut or lung. IMPORTANT NOTE: Working solution of sodium bisulfite must be used the same day as it is prepared.
5. Add 400 μl of 25 mg/ml proteinase K to 40 ml of 2× SSC, pH 7.0. Incubate slides in this proteinase K working solution 10 to 30 min at 45°C. IMPORTANT NOTE: Working solution of proteinase K must be used the same day as it is prepared. The proteinase K concentration and treatment time must be determined empirically. Start with 10 min incubation time. Alternatively, a commercially available pretreatment kit (i.e., Abbott Molecular) can be used according to the manufacturer’s instructions.
6. Rinse slides in three changes of 2× SSC, pH 7.0 for 1 min each at room temperature.
Dehydrate the sample 7. Dehydrate slides in 70%, 80%, 90%, and 100% ethanol by immersing for 2 min each at room temperature. Air dry slides 8. Stain slides with propidium iodide/Antifade (apply 10 μl and cover with a 22 × 22mm glass coverslip) and evaluate using fluorescence microscopy (UNIT 4.4; McNamara et al., 2005) to determine the degree of digestion. Over-digestion will cause loss of nuclear borders, may give a “ghostly” appearance of nuclei, and ultimately lead to a loss of material altogether. Persistent green autofluorescence and poor propidium iodide staining indicate that the tissue is underdigested. In this case, slides should be transferred to 2× SSC, pH 7.0, for several minutes before repeating steps 4 and 5.
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22 X 22–mm glass coverslip
sample nuclei
rubber cement
glass slide
Figure 8.8.1 Placement of sample nuclei on glass slide to allow two separate hybridizations, with two probes or two sets of probes, to be performed simultaneously.
Prepare probe 9. Prepare DNA probes as described in UNIT 4.3 (Knoll and Lichter, 2005). Preparation of DNA probes including labeling, use of Cot1 DNA, along with concentration guidelines and methods for detection of the hybridized probe are described in UNIT 4.3 (Knoll and Lichter, 2005). For commercial probes, follow manufacturer’s instruction. In general, the concentration of probe depends on the size and complexity of the target sequence. Five to twenty ng of probe/μl of hybridization buffer usually works well (e.g., 5 to 10 ng/μl for whole chromosome paint or cosmid probes, 15 to 20 ng/μl for P1, BAC, YAC probes). Ten μl of hybridization mixture (i.e., hybridization buffer + probe) is sufficient to cover the area under 22 × 22-mm coverslip. IMPORTANT NOTE: Store probes at –20°C and protect directly labeled probes (e.g., Spectrum Orange, Spectrum Green) from light. Prolonged storage of the hybridization mixture at room temperature prior to application to the sample should be avoided.
Hybridize probe to the sample 10. Pipet 10 μl of hybridization mixture onto target area, cover with glass coverslip, and seal with rubber cement (Fig. 8.8.1). If using two different probes on the same slide, apply one probe, coverslip (usually a 22 × 22-mm coverslip works best), and sealant before applying the second probe to prevent the probes from mixing (see Fig. 8.8.1).
11. Denature probe and target DNA at 95°C for 10 min. Depending on the number of slides, it may be necessary to heat the oven up to 100°C to maintain 95°C. In general, a 10-min denaturation time works well. However, if the nuclei are fragile due to some unknown factor of tissue fixation or storage, denaturation times can be shortened to 5 min or temperatures decreased to as low as 75°C with minimal loss of final hybridization signal intensity. Alternatively, a slide warmer (e.g., HYBrite) can be used for denaturation.
12. Incubate slides in a humidified chamber at 37°C or 42°C overnight to allow hybridization to occur.
Wash off excess probe 13. Remove coverslips and wash slides, in 50-ml glass Coplin jars, as follows: a. Once in 0.4× SSC pH 7.0/0.3% (v/v) Tween 20 at 73°C for 2 min. b. Once in 2× SSC pH 7.0/0.1% (v/v) Tween 20 at room temperature for 1 min. Preparation of Cells from Fixed Tissue for Use in FISH Experiments
This wash procedure usually yields preparation with a low signal-to-noise ratio. If an excess background is observed, repeat wash and more frequent agitation of slides is recommended. For probes that yield a relatively weak hybridization signal, lower wash
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stringency is recommended (e.g., 50% formamide/2× SSC, 2× SSC at 39°C or 42°C, UNIT 4.3; Knoll and Lichter, 2005).
14. Transfer slides to Coplin jar containing 1× PBD at room temperature. At this point the slides can be stored in 1× PBD over night at 4°C if necessary. The manual procedure described above can be automated by using the ThermoBrite Elite FISH Auto Slide Prep (Leica Biosystems).
Detect hybridized probe Detection and amplification of hybridization signals of biotin- or digoxigenin-labeled probes is described in detail in UNIT 4.3 (Knoll and Lichter, 2005). No detection is required for directly labeled probes. 15. Counterstain with DAPI II/Antifade solution (10 μl) and cover with a 24 × 50-mm no.1 glass coverslip. 16. Examine slides with a fluorescence microscope (UNIT 4.4; McNamara et al., 2005) using the appropriate filter set. Record the hybridization pattern or number of hybridization signals per nucleus.
PREPARATION OF SINGLE-CELL SUSPENSION FROM FORMALIN-FIXED, PARAFFIN-EMBEDDED TISSUE FOR FISH
BASIC PROTOCOL 2
This protocol requires 50-μm tissue sections cut from selected areas of paraffin blocks. The tissue is deparaffinized, rehydrated, and disaggregated using proteolytic enzymes. The protocol is based on a method described by Kuchinka et al. (1995), with modifications as described below.
Materials Paraffin-embedded tissue of interest Silanized glass (precleaned), or positively charged, microscope slides (see recipe) Xylene Ethanol Hanks’ balanced salt solution (HBSS; APPENDIX 2D) 1 mg/ml collagenase XI solution (Sigma-Aldrich) 0.05% trypsin/EDTA (Invitrogen) Sterile H2 O Microtome 15-ml glass conical centrifuge tubes Centrifuge (e.g., Marathon 3000, Fisher Scientific) Vortex 50°C oven (or slide warmer; e.g., HYBrite, Abbott Molecular, Leica Biosystems) Additional reagents and equipment for probe hybridization (see Basic Protocol 1) Deparaffinize and rehydrate the sample 1. Using a microtome, cut two to five 50-μm sections and place in 15-ml glass conical centrifuge tubes. The number of tissue sections needed depends upon both the size and cellularity of the material; for most types of tissue, two 1 × 1-cm sections should yield enough nuclei to perform hybridization with 10 probes.
2. Add 5 ml xylene and let stand 5 min, agitate occasionally, then centrifuge 30 to 60 sec at 287 × g, room temperature. Discard supernatant.
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The number of specimens that can be disaggregated at one time is limited by both the number of tubes that the centrifuge holds and manual constraints later in the procedure. In general, it is cumbersome to handle >10 specimens per run.
3. Repeat step 2 using the following solutions in turn:
5 ml xylene 5 ml 100% ethanol 5 ml 100% ethanol 5 ml 80% ethanol 5 ml 50% ethanol 5 ml sterile H2 O Leave in water overnight at 4°C At this point visual inspection of the tube should show no evidence of white waxy paraffin flecks. If a few fragments of paraffin are observed, they can be removed manually. If paraffin fragments cannot be removed, the tissue should be dehydrated by repeating the wash and centrifuge steps described in steps 2 and 3 above but using the solvents in reverse order (i.e., 50% ethanol, 80% ethanol, 100% ethanol, 100% ethanol, xylene, xylene). Increasing xylene treatment time with occasional agitation is recommended. Repeat step 3 to rehydrate the tissue.
4. Centrifuge for 30 to 60 sec at 287 × g, room temperature. Discard supernatant. 5. Wash 1× in HBSS. Centrifuge for 30 to 60 sec at 287 × g, room temperature, and discard supernatant.
Extract nuclei 6. Add 1 ml collagenase XI solution (1 mg/ml) and incubate at 37°C for 2 hr. Add 9 ml HBSS. Centrifuge for 30 to 60 sec at 287 × g, room temperature, then discard supernatant. 7. Add 5 ml of 0.05% trypsin/EDTA; incubate at 37°C for 1 hr or longer. Centrifuge for 30 to 60 sec at 287 × g, room temperature, and discard supernatant. For previously untested tissue start with 30 min of trypsin treatment and check for disaggregation. A 1-hr incubation usually gives good results.
8. Wash tissue with 5 ml HBSS once. Centrifuge for 30 to 60 sec at 287 × g, room temperature, discard supernatant and then add 1 ml HBSS. Vortex vigorously and allow large pieces of tissue to settle (1 to 2 min). 9. Drop cell suspension on slide using a Pasteur pipet. Single cells should be visible by phase-contrast microscopy. Check cell concentration and dilute if necessary by adding more HBSS.
Prepare slide 10. Place a drop of cell suspension on one or two areas of a glass slide (Fig. 8.8.1), bake for 2 hr or overnight at 50°C. Transfer remaining cell suspension to a microcentrifuge tube and store at 4°C. For longer storage remove supernatant and resuspend pellet in 70% ethanol. Store at −80°C.
Preparation of Cells from Fixed Tissue for Use in FISH Experiments
Dehydrate the sample 11. Dehydrate slides by immersing in 70%, 80%, 90%, and 100% ethanol for 2 min each at room temperature and allow to air dry. The slides are ready to use for hybridization, or they can be stored in a dry chamber for several weeks. If stored, dehydrate the slide again before use.
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Hybridize probe to the sample 12. Hybridize, wash and examine slides as described in Basic Protocol 1, steps 10 to 14. REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2D; for suppliers, see SUPPLIERS APPENDIX.
Silanized glass microscope slides Prepare a 2% (v/v) silanization solution by mixing 2 ml of 3aminopropyltriethoxysilane (Sigma-Aldrich) and 98 ml acetone. Dip slides into silanization solution for 20 sec. Wash in two changes of acetone, 15 sec each, then air dry. Store slides indefinitely at room temperature.
COMMENTARY Background Information Routine cytogenetics based on chromosomal banding techniques has been successful in correlating constitutional or acquired karyotype abnormalities with clinical diagnosis, prognosis, and, in some cases, therapeutic response. However, this requires a source of viable, mitotic cells with good chromosome morphology and usually involves a relatively long turn-around time. The advent of molecular cytogenetics, with development and optimization of fluorescence in situ hybridization (FISH), brought considerable progress especially in cancer cytogenetics, and at present the technique is widely used for both research and clinical studies (see UNIT 4.3; Knoll and Lichter, 2005). DNA segments of interest serve as probes after a fluorescent tag or a reporter molecule is attached. Under proper hybridization conditions the probe recognizes and binds to the homologous sequences in target DNA. After hybridization, the target homologous regions are recognized and evaluated by using fluorescence microscopy. The technology is simple and robust and its significant contribution, especially to cancer cytogenetics, relies on its applicability to interphase cells. Thanks to the Human Genome Project, scores of genomic DNA fragments were fully characterized and mapped to specific chromosomes and chromosome regions. This allowed creation of YAC, BAC, cosmid or plasmid libraries, which serve as an excellent source of specific DNA probes in addition to those previously employed probes comprised of alpha, beta or classical satellite DNAs. Most FISH studies are performed on cells from fresh or frozen tissues. However, it has been well documented that formalin-fixed, paraffin-embedded tissue can provide a viable alternative (Schofield and Fletcher, 1992;
Kuchinka et al., 1995; Di Francesco et al., 2000; Pickering et al., 2001; Gelpi et al., 2003) when fresh tissue is not available. The use of fixed tissues increases the possibility of analyzing large number of archival cases of rare anomalies. FISH analysis of formalin-fixed, paraffinembedded tissue can be performed on unstained histological tissue sections or on disaggregated, intact nuclei. Unstained sections should be used when preservation of tissue morphology is necessary. A limitation of this technique is that it results in many incomplete nuclei, which can lead to a loss of some chromosomal material and subsequently to underestimation of chromosome copy number. Interpretation of results requires statistical analysis of the FISH signals observed in both normal tissue and the clinical specimen and establishing laboratory cut-offs for aneuploidy evaluation (Qian et al., 1996) or target-toreference probe ratio in cases tested for a loss of a target allele (Gelpi et al., 2003). However, this method allows precise histopathological correlation of multiple foci of normal cells, premalignant lesions, and tumor cells within a single specimen, including study of intratumor heterogeneity. The second approach involves extraction of intact nuclei from 50-μm tissue sections. The method eliminates the problem of loss of chromosomal material due to sectioning of nuclei and can be used to evaluate both numerical and structural chromosome abnormalities (Kuchinka et al., 1995; Qian et al., 1996; Pickering et al., 2001). The main disadvantage is loss of the spatial relationships of the cells and possible admixture of normal stromal cells. This problem can be circumvented by using hematoxylin and eosin (H & E) stained sections and careful evaluation of
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selected paraffin blocks to identify the best areas for sampling (e.g., highest percentage of tumor cells, fetal parts of placenta, areas free of necrosis; DiFrancesco et al., 2001; Pickering et al., 2001; Gelpi et al., 2003). FISH analysis of isolated nuclei does not require special training in pathology, and counting/evaluation of fluorescence signals is easier on isolated nuclei than on tissue sections. Finally, large-scale high-throughput adaptation of this technique is feasible.
Critical Parameters and Troubleshooting
Preparation of Cells from Fixed Tissue for Use in FISH Experiments
Tissue fixation Optimally, tissue samples should be fixed in neutral-buffered formalin or paraformaldehyde for 24 to 40 hr. Parameters such as fixation time, block size, and the embedding process all can affect FISH analysis. Prolonged fixation time may impair access of probe to the target DNA due to excessive cross-linking between nuclear proteins and between proteins and DNA. Tissue quality is important; therefore delay in fixation time, leading to decay and autolysis of the tissue, may cause weak or absent hybridization signals. Preparation of paraffin sections from specimens containing bone or calcified tissue areas (e.g., dystrophic calcification, metastatic calcification) requires thorough fixation to protect cells and fibrous elements of the tissue from damage caused by decalcifying agents (e.g., acids, chelating agents) during the subsequent decalcifying process. For fixation, buffered formalin is preferable; however, in some cases where preservation of a specific tissue (e.g., bone marrow) is important, alternative fixatives such as B5 or Bouin’s are used. Commercial suppliers of DNA probes consider some fixatives including B5, Zenker’s, Zamboni’s, Bouin’s, and mercuric chloride as incompatible with standard FISH protocols. Prolonged treatment with decalcifying agents can cause DNA degradation; therefore, FISH analysis on routinely prepared paraffin sections from specimens containing bone or calcified tissue areas might be unsuccessful. If FISH testing is needed on tissue sections from such specimens, prolonged treatment with decalcifying agents and/or application of fixatives incompatible with FISH must be avoided, and the protocol(s) will require modifications. For example, a limited acid decalcification with 5% formic acid (Brown et al., 2002), ultrasonic decalcification (Reineke et al., 2006), or alternatively, closely monitored decalcification us-
ing EDTA (Zustin et al., 2009) protocols can be applied, and a method described by Schurter et al. (2002) can be used when FISH is performed following use of one of the FISH-incompatible fixatives. Tissue pretreatment/digestion Removal of proteins via pretreatment with sodium bisulfite solution and digestion with proteolytic enzymes (e.g., proteinase K, pepsin) or other commercial pretreatment solutions (from, e.g., Vysis, Inc., MP Biochemicals) reduces autofluorescence, enhances accessibility of target DNA, and may lead to improvement of hybridization signal intensity. Concentration of proteinase K or pepsin is critical because for both, insufficient or excess digestion will affect hybridization. Underdigestion leads to autofluorescence; overdigestion will compromise nuclear morphology and/or cause loss of the tissue. Incubation time can vary depending on tissue type and length of fixation time. Therefore, specific treatment times must be determined empirically from case to case or tissue to tissue. For in vitro diagnostic commercial products, the manufacturer’s protocols and recommendations should be followed. During preparation of the single-cell suspensions (Basic Protocol 2) potential problems include insufficient numbers or poor morphology of nuclei. Insufficient numbers of nuclei can be a result of a hypocellular tissue sample or incomplete tissue digestion. The specimen should be re-evaluated and additional sections from the same or different block should be obtained to repeat the experiment. If hypocellularity is not a problem, then digestion time should be increased. Poor morphology of nuclei or excessive nuclear/cellular debris can be a result of poor sample quality (necrosis) or over-digestion. Re-evaluation of the tissue block and/or decreased digestion time should be considered. In situ hybridization For problems related to in situ hybridization (background, cross-hybridization, etc.) see UNIT 4.3 (Knoll and Lichter, 2005). Efficiency of hybridization and hybridization signals can be affected by the quality of tissue sample, fixation time, and type of fixative. To some degree, such problems can be solved by modifying the standard protocol (Hyytinen et al., 1994; Schurter et al., 2002); however, in some cases they may remain intractable. Increasing the denaturation time can be considered.
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Anticipated Results Hybridization efficiency of over 90% can be achieved for nuclei prepared from tissue samples collected at the time of surgery and processed appropriately. For samples collected at autopsy, hybridization results are more variable, and in some cases the nuclei may fail to hybridize. To interpret FISH results using Basic Protocol 1, an adequate number of nuclei must be evaluated before performing appropriate statistical analysis such as a Student’s t-test and/or chi-square test (Weremowicz et al., 1994; Qian et al., 1996; Le Maitre et al., 2001) to compare test results to control probes, or normal controls, and to establish laboratory cut-offs. Evaluation of 30 to 50 nuclei from single-cell suspensions (Basic Protocol 2) generally gives an interpretable result (S. Weremowicz, unpub. observ.).
Time Considerations Both protocols are labor intensive and time consuming. FISH results on tissue sections (Basic Protocol 1) can be available in 72 hr and on disaggregated cells (Basic Protocol 2) in 96 hr. However, the opportunity to evaluate archival material and to acquire valuable clinical information, otherwise not obtainable, far outweighs the labor requirement. To decrease turn-around time, the FISH procedure on tissue sections might be modified by applying a microwave irradiation method (Kitayma et al., 2000; Ridderstrale et al., 2005).
Literature Cited Brown, R.S.D, Edwards, J., Bartlett, J.W., Jones, C., and Dogan, A. 2002. Routine acid decalcification of bone marrow samples can preserve DNA for FISH and CGH studies in metastatic prostate cancer. J. Histochem. Cytochem. 50:113-115. Di Francesco, L.M., Murthy, S.K., Luider, J., and Demetrick, D.J. 2000. Laser capture microdissection-guided fluorescence in situ hybridization and flow cytometric cell cycle analysis of purified nuclei from paraffin sections. Mod. Pathol. 13:705-711. Emmerich, P., Jauch, A., Hofmann, M.-C., Cremer, T., and Walt, H. 1989. Interphase cytogenetics in paraffin embedded sections from testicular germ cell tumor xenografts and in corresponding cultured cells. Lab. Invest. 61:235-242. Gelpi, E., Ambros, I.M., Birner, P., Leugmayr, A., Drlicek, M., Fischer, I., Kleinert, R., Maier, H., Huemer, M., Gatterbauer, B., Anton, J., Rossler, K., Budka, H., Ambros, P.F., and Hainfellner, J.A. 2003. Fluorescent in situ hybridization on isolated tumor cell nuclei: A sensitive method for 1p and 19q deletion analysis in paraffinembedded oligodendrial tumor specimens. Mod. Pathol. 16:708-715.
Hopman, A.H.N., van Hooren, E., van de Kaa, C.A., Vooijs, P.G.P., and Ramaekers, F.C.S. 1991. Detection of numerical chromosome aberrations using in situ hybridization in paraffin sections of routinely processed bladder cancers. Mod. Pathol. 4:503-513. Hyytinen, E., Visakorpi, T., Kallioniemi, A., Kallioniemi, O.-P., and Isola, J.J. 1994. Improved technique for analysis of formalin-fixed, paraffin-embedded tumors by fluorescence in situ hybridization. Cytometry 16:93-99. Kitayama, Y., Igarashi, H., and Sugimura, H. 2000. Initial intermittent microwave irradiation for fluorescent in situ hybridization analysis in paraffin-embedded tissue sections of gastrointestinal neoplasia. Lab. Invest. 80:779781. Knoll, J.H.M. and Lichter, P. 2005. In situ hybridization to metaphase chromosomes and interphase nuclei. Curr. Protoc. Hum. Genet. 45:4.3.1-4.3.31. Kuchinka, B.D., Kalousek, D.K., Lomax, B.L., Harrison, K.J., and Barrett, I.J. 1995. Interphase cytogenetic analysis of single cell suspensions prepared from previously formalin-fixed and paraffin-embedded tissues. Mod. Pathol. 8:183186. Le Maitre, C.L., Byers, R.J., Liu Yin, J.A., Hoyland, J.A., and Freemont, A.J. 2001. Dual colour FISH in paraffin bone trepines for identification of numerical and structural chromosome abnormalities in acute myeloid leukemia and myelodysplasia. J. Clin. Pathol. 54:730-733. McNamara, G., Difilippantonio, M.J. and Ried, T. 2005. Microscopy and image analysis. Curr. Protoc. Hum. Genet. 46:4.4.1-4.4.34. Pickering, D.L., Nelson, M., Chan, W.C., Huang, J.Z., Dave, B.J., and Sanger, W.G. 2001. Paraffin tissue core sectioning: An improved technique for whole nuclei extraction and interphase FISH. J. Assoc. Gen. Tech. 27:38-40. Qian, J., Bostwik, D.G., Takahashi, S., Borell, T.J., Brown, J.A., Lieber, M.M., and Jenkins, R.B. 1996. Comparison of fluorescence in situ hybridization analysis of isolated nuclei and routine histological sections from paraffinembedded prostatic adenocarcinoma specimens. Am. J. Pathol. 49:1193-1199. Reineke, T., Jenni, B., Abdou, M.-T., Frigerio, S., Zubler, P., Moch, H., and Tinguely, M. 2006. Ultrasonic decalcification offers new perspectives for rapid FISH, DNA, and RT-PCR analysis in bone marrow trephines. Am. J. Surg. Pathol. 30:892-896. Ridderstrale, K.K., Grushko, T.A., Kim, H.-J., and Olopade, O.I. 2005. Single-day FISH procedure for paraffin embedded tissue sections using a microwave oven. BioTechniques 39:316320. Schofield, D.E. and Fletcher, J.A. 1992. Trisomy 12 in pediatric granulosa-stromal cell tumors: Demonstration by a modified method of fluorescence in-situ hybridization on paraffinembedded material. Am. J. Pathol. 141:12651269.
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Schurter, M.J., LeBrun, D.P., and Harrison, K.J. 2002. Improved technique for fluorescence in situ hybridisation analysis of isolated nuclei from archival, B5 or formalin fixed, paraffin wax embedded tissue. J. Clin. Pathol. Mol. Pathol. 55:121-124. Tkachuk, D.C., Westbrook, C., Andreef, M., Donlon, T.A., Clearly, M.L., Suryanaryan, K., Homge, M., Redner, A., Gray, J., and Pinkel, D. 1990. Detection of bcr-abl fusion in chronic myelogenous leukemia by in situ hybridization. Science 250:559-562. Walt, H., Emmerich, P., Cremer, T., Hofmann, M.-C., and Bannwart, F. 1989. Supernumerary chromosome 1 in interphase nuclei of atypical germ cells in paraffin-embedded hu-
man seminiferous tubules. Lab. Invest. 61:527531. Weremowicz, S., Kozakewich, H.P., Haber, D., Park, S., Morton, C.C., and Fletcher, J.A. 1994. Identification of genetically aberrant cell lineages in Wilms’ tumors. Genes Chromosomes Cancer 10:40-48. Zeller, R. 1989. Fixation, embedding, and sectioning of tissues, embryos, and single cells. Curr. Protoc. Mol. Biol. 7:14.1.1-14.1.8. Zustin, J., Baddin, K., Tsourlakis, M.C., Burandt, E., Mirlacher, M., Jaenicke, F., Izbicki, J., Reuther, W., Rueger, J.M., Bokemeyer, C., Simon, R., and Sauter, G. 2009. HER2/neu analysis in breast cancer bone metastases. J. Clin. Pathol. 62:542-546.
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