C 1990 Wiley-Liss, Inc.
Cytometry 11:126-131 (1990)
Multiple Fluorescence In Situ Hybridization P.M. Nederlof, S. van der Flier, J. Wiegant, A.K. Raap, H.J. Tanke, J.S. Ploem, and M. van der Ploeg Sylvius Laboratories, Department of Cytochemistry and Cytometry, University of Leiden, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Received for publication May 23, 1989; accepted J u n e 17, 1989
A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets
Chromosome banding techniques are widely applied for the identification of numerical and/or structural chromosome aberrations in tumor and prenatal diagnosis. The interpretation of the banding patterns requires skilled technicians; it is often complicated by imperfect banding, chromosome condensation, and limited numbers of metaphases; and i t is extremely difficult in case of highly aneuploid tumors with extensive structural changes. An alternative method for the detection of numerical chromosome aberrations is the in situ hybridization technique with chromosome specific repetitive probes, which also has the advantage that interphase nuclei can be analysed (3,5,8,15). One has to keep in mind that chromosome specific satellite probes only give information about the centromeric region of the chromosome, and therefore do not allow detection of (structural) chromosome aberrations which involve other parts of that chromosome. Recently, however, probes have been developed from chromosome specific libraries which can be used for overall staining of specific individual chromosomes by using competitor total genomic DNA to exclude dispersed repetitive sequences from participating in the in situ hybridization (2,161. As a n alternative to this type of probe “cocktail,” DNA from somatic cell hybrids containing specific chromosomes (or fragments) can be used (11).The combination of specific integral cytochemical chromosome staining procedure with chromosome specific satellite probes allows, in principle, detection of considerably more numerical and structural aberrations (translocations).
simultaneously is important for prenatal diagnosis and the detection of numerical andlor structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique. Key terms: Chromosomal aberrations, prenatal diagnosis, oncology, fluorescence ratio imaging
The possibility of detecting multiple sequences simultaneously is very important, since i t allows analysis of nuclei for different aberrations a t the same time. The number of simultaneously detectable target sequences by fluorescence in situ hybridization is determined by the number of specific probes, different labeling techniques, independent (immunological) detection methods, and microscopically detectable labels. With the large number of probe haptenization procedures available, it appears that currently the maximum number of simultaneously detectable probes is limited by the number of immunofluorophores available. Although in fluorescence microscopy and evaluation by the naked human eye the number of spectrally separated fluorochromes is practically limited to three colors (blue (AMCA); green (FITC); red (TRITC, Texas Red)), we show in this paper that the number of simultaneously detectable probes can be increased to more than three. This is achieved by combining double haptenized probes and three fluorochromes in such a way that in principle up to seven targets can be detected (Table 1). Preliminary results, using fluorescence digital image microscopy (FDIM) (l),indicate that the fluorescence intensity ratios of double haptenized probes
Address reprint requests to Dr. P.M. Nederlof, Sylvius Laboratories, Department of Cytochemistry, University of Leiden 2333 AL Leiden. The Netherlands.
are fairly constant. Thus by using probes with different hapten ratios and fluorescence ratio imaging, the number of simultaneously detectable probes can be increased even further. The benefit of multiple in situ hybridization for cytogenetic analysis lies primarily in the fact that automated and more objective identification of chromosome (segments) is feasible. Furthermore, multiple hybridizations with fluorescence ratio imaging in combination with high quality image representation using artificial color may form the basis of a n in situ hybridization chromosome banding procedure.
MATERIALS AND METHODS Preparation of the Microscopical Slides Metaphase preparations were obtained from phytohaemagglutinin stimulated human peripheral blood lymphocytes from a normal male (46, XY), which were treated for 1 h with Colcemid (0.5 pgiml). After centrifugation cells were resuspended in a prewarmed (37°C) swelling buffer containing 50 mM KC1, 10 mM MgSO,, 3 mM DTT, and 5 mM K2HP04 pH 8.0. After incubation for 30 min a t 37°C cells were fixed with four changes of methanoliacetic acid (3:l viv). Cell suspensions were stored a t -20°C. Cells were centrifuged on cleaned (ethanoliether, 1/ 1) slides, allowed to air dry (overnight), washed with phosphate-buffered saline (PBS: 0.15 m NaC1, 10 mM Na phosphate, pH 7.21, and gradually dehydrated with ethanol. Before use, slides were treated with 100 pgiml RNase A in 2 x SSC (0.3M NaC1,30 mM Na citrate, pH 7.2) under a coverslip for 60 min at 37"C, with proteinase K (0.1 ygiml in 20 mM Tris-HC1, 2 mM CaCl,, pH 7.4), for 7.5 min a t 37°C and were post-fixed with 4% (para)formaldehyde (in PBS, 50 mM MgC12)for 10 min, dehydrated, and kept a t room temperature until used.
DNA Probes and Labeling Human alphoid or simple satellite probes specific for chromosomes #1 (pUC1.77) (4),#7 (pa7tl) (19), #15 (D15Z1) (7), and #17 (p17H8) (18) were labeled with biotin (bio) according to Langer et al. (13),with amino acetylf luorene (AAF) according to Landegent et al. (121, or with the Chemiprobe kit (CP) (17) (FMC Bioproducts, generous gift of Amstelstad BV, Zwanenburg, NL) according to the manufacturer's instructions. In case of double haptenization with AAF and biotin, the (non-sonified) probes were first labeled with AAF, precipitated, and dissolved in a nick-translation buffer for labeling with bio-11-dUTP. For the combination Chemiprobeibiotin, probes were first labeled with CP.
Table 1 Scheme for Multiple Hybridization Combining Biotin, AAF, and Chemiprobe in Single-, Double-, and Triple-Probe Labeling
Label bio AAF CP biolAAF bioiCP AAFICP bioiAAFiCP
Red TRITC
Color signal Green FITC
+~
+ ++
Blue AMCA ~
+ ~
-
+ + +
-
+ + + +
In Situ Hybridization For each hybridization the labeled probe(s) was mixed in a hybridization buffer containing 60% deionized formamide, 2 x SSC, carrier DNA (50 times excess of salmon sperm DNA and yeast RNA). Per hybridization, 7 p1 of probe mixture is used (containing 20 ng for the probes: p#7-bio, p#15-AAF, p#l7--Cp, and 10 ng for p#l-AAFibio). The probes and target were denatured together under a coverslip (18x 18 mm) a t 80°C for 5 rnin in a n incubator. Hybridization was performed in a moist chamber for 16 h a t 37°C. Slides were then washed a t room temperature two times 5 min with 60% formamide, 2 x SSC, and two times 10 min with 2 x SSC. Detection After a wash with PBSiTween-20 for 5 min, slides were incubated a t 37°C for 30 min with 100 ~1 PBSi Tween-20 with 5% normal goat serum under a 24 x 50 mm coverslip. All immunological incubations were performed in PBS containing 0.05% Tween-20, 5% normal goat serum, and the appropriate dilution of antisera. Incubations were for 45 min a t 37°C in a moist chamber. After each incubation the slides were washed three times with PBS/O.O5%,Tween-20. For the detection of the double labeled probe ( p # l AAFibio) in the first layer a mouse anti-AAF monoclonal antibody (diluted 1500, kindly provided by Dr. R.A. Baan, TNO, Medical Biological Laboratories, Rijswijk) in combination with TRITC conjugated avidin (1:4000, Zymed, USA) was used. The second step contained FITC conjugated rabbit anti-mouse IgG, (1:800, Vector, USA). For the quadruple hybridization, the first layer consisted of a mouse monoclonal anti-Chemiprobe (from the Chemiprobe-kit) (1:800), a rabbit anti-AAF polyclonal antiserum (1500, R.A. Baan), and TRITC conjugated avidin (1:4,000).The second layer contained a n AMCA conjugated goat anti-mouse (1:200, Sigma, USA), prepared a s described by Khalfan e t al. (10) USing N-hydroxysuccimide-AMCA (Biocarb, Lund, Sweden) and a FITC conjugated goat anti-rabbit antibody (1:800, Sigma, USA).
MU1,TIPLE FLITOHESC'ENCE IN SITIT HYKI:IDIZATION
129
FIG 3. Quadruple hybridization p#l-bioiAAF iTRITC/FITC), p#7-bio (TRITC), p#15-AAF iFITC), p#17-CP (AMCA),on a n interphase nucleus lymphocyte.
Microscopy
photomicrographs were taken with a Leitz ~ i microscope with a Vario-orthomat, equipped for FITC, TRITC, and AMCA fluorescence (14) using a 3~ color slide film. E~~~~~~~times were about 1-2 min for FITC, 30 s for AMCA, and 90 s for TRITC.
RESULTS AND DISCUSSION The different haptens used for the labeling of DNA probes bind to different nucleotides; it is possible, therefore, to double label one probe with two (or more) different haptens, which can be visualised in different colors. Biotin labeling was performed by nick-translation with bio-11-dUTP (the biotin is coupled to the C5 position of the uridine base); 2-acetyl aminofluorene
~ _ _ _ _
F!G 1. Single hybridization with a double labeled probe p#l-bio/ AAF (FITCITRITC) on lymphocyte metaphase chromosomes, DAPI counterstained. A. Double exposure with blue excitation (FITC) and UV excitation (DAPI). B: Double exposure with green excitation (TRITC) and UV excitation. FIG 2 . Triple hybridization p#l-bio/CP (FITC/TRITC), p#7-bio (FITC), p#17-CP (TRITC), on metaphase chromosomes, DAPI counterstained. A Double exposure with blue and UV excitation for, respectively, FITC and DAPI. B: Double exposure with green and UV excitation for TRITC and DAPI.
(AAF) is bound covalently to the C8 position of guanosine and the~ Chemiprobe kit labels the C6 ~ residues; l ~ Position of cytosine residues with a sulfonate group. Although modification with biotin and AAF alters the stability of the hybrid formation since it affects the base pairing, the double labeled probes retained their chromosome specificity under the hybridization conditions applied (60% formamide, 2 x SSC, 37°C). For double labeling with AAF and biotin best results were obtained when the AAF labeling was performed prior to the biotin labeling. The nick-translation reaction time was then reduced to 1h incubation (normally 2 h). Also double labeled probes combining AAF and Hg, or CP and biotin, were used, giving similar results (not shown). Instead of a double labeled probe, i t is also possible to use a mixture of two different single labeled probes (results not shown). In Figure 1 the results of a hybridization of a double labeled probe for chromosome #1 with AAF and biotin are given. On the metaphase chromosomes the AAF hapten is visualised in red (TRITC) and the biotin label in green (FITC). The FITC signal is very intense, and also some minor binding sites are visible. The fact that the green and red fluorescence signal are not exactly on the same position is caused by the need for changing filter blocks for FITC and TRITC, thereby slightly changing the orientation of the dichroic mirror. The availability of new filter blocks consisting of one excitation filter and dichroic mirror which allow simultaneous detection of FITC and Texas Red will eliminate this phenomenon.
By applying these double labeled probes in combination with only two single labeled probes it is possible to perform tripie hybridizations using only two fluorochromes (FITC: green, and TRlTC: red); this has the advantage that DAPI (blue) can be used a s total DNA counterstain. Probe p#l-biolCP was combined with p#7-bio and p# 17-CP in a triple hybridization experiment on human lymphocytes (Fig. a), showing chromosome #1 in both green and red, chromosome #7 in green, and chromosome #17 in red. Such a double labeled probe can also be used in a quadruple-hybridization, when used in combination with three single labeled probes. For the simultaneous detection of four targets in lymphocyte nuclei four different probes were used, the double labeled p#l-AAFI bio probe and three single labeled probes: p#7-bio, p#15-AAF and p#17-CP. In Figure 3 the results of this fourfold hybridization are shown: chromosome #1 is visible in both green and red, chromosome #7 in red, chromosome #15 in green, and finally chromosome #17 in blue. The number of different probes used in one in situ hybridization can in principle already be extended from four to seven by using three single labeled probes, three double labeled probes, and one triple labeled probe. Introduction of infra-red emitting cyanin dyes ( 6 ) can increase the multiplicity of in situ hybridizations even further. Visual interpretation will be complex, however, and impossible for the infra-red dyes. Therefore, digitizing of fluorescent images and visualization with pseudo-colors will be required. Fluorescence digital imaging microscopy using sensitive (charge-coupled device) cameras is ideally suited for this purpose (I).Preliminary measurements, using digital fluorescence imaging microscopy with an intensified video camera (Hamamatsu VIM system, C1966-20, Hersching, West Germany), so far show that the intranuclear intensities of in situ hybridization signals obtained with chromosome specific satellite probes are fairly constant. The internuclear variation is however considerable, probably due to differences in hybridization efficiencies. Internal standards and therefore, additional hybridizations will be necessary to correct for these internuclear differences. Furthermore, measurement of the FITC and TRITC spot intensities of double labeled (chromosome specific satellite) probes revealed that the FITCiTRITC intensity ratio is fairly constant (S.D. < 10%). For fast automated screening for chromosomal aberrations for prenatal diagnosis, the three autosomal chromosomes #13, #18, and #21 and the sex chromosomes are clinically the most important; therefore, the possibility to perform a fivefold hybridization is of great interest. When the hapten ratio on the probe can be controlled, measurement of the FITCITRITC ratio may allow identification of different probes on basis of their difference in hapten ratios. This will increase the number of detectable probes even more by using only three
colors (comparable to the use of two-color immunofluorescence in flow cytometry; ref. 9). This would create the possibility to detect in a n automated fashion multiple chromosomes by fluorescence ratio imaging, and may also be the basis for a n in situ hybridization banding technique using differently (double) labeled probe cocktails from chromosome (segment) specific libraries.
ACKNOWLEDGMENTS The authors thank Hamamatsu Photonics Europa GMBH (Herrsching, West Germany) for placing at our disposal the Hamamatsu VIM system.
LITERATURE CITED 1. Arndt-Jovin DJ, Robert-Nicoud M, Kaufman SJ, Jovin TM: Fluorescence digital imaging microscopy (DIM) in cell biology. Science 230:247, 1985. 2 . Cremer T, Lichter P, Borden J, Ward DC, Manuelidis L: Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes. Hum Genet 80235-246, 1988. 3 Cremer 'T,Tessin D, Hopman AHN, Manuelidis L: Rapid interphase and metaphase assessment of specific chromosomal changes in neuroectodermal tumor cells by in situ hybridization with chemically modified DNA probes. Exp Cell Res 176:199220,1989. 4 Devilee P, Cremer T, Slagboom P, Bakker E, Scholl HP, Hager HD, Stevenson AFG, Cornelisse CJ, Pearson PL: Sequence heterogenei ty within the alphoid repetitive DNA family. Cytogenet Cell Genet 41:193-201. 1986. 5 Devilee P , Thierry RF, Kievits T, Tollurj R, Hopman AHN, Willard HF, Pearson PL, Cornelisse CJ: Detection of chromosome aneuploidy in interphase nuclei from human primary breast tumors using chromosome specific repetitive DNA probes. Cancer Res 48:5'325-5830, 1988. 6 Ernst LA, Gupta RK, Mujumdar RB, Waggoner AS: Cyanine dye labeling reagents for sulfhydryl groups. Cytometry 10:3-10, 1989. 7 Higgins MJ, Wang H, Shtromas I, Haliotis T, Roder JC, Holden J J A , White BN: Organization of a repetitive human 1.8kb KpnI sequence localized in the heterochromatin of chromosome 15. Chromosoma 93:77-86, 1985. 8 Hopinan AHN, Kamaekers FCS, Raap AK, Beck JLM, Devilee P, van der Ploeg M, Vooijs GP: In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors. Histochemistry 89:307-316, 1988. 9 Horan PK, Slezak SE, Poste G: Improved flow cytometric analysis of leukocyte subsets: Simultaneous identification of five cell subsets using two color immunofluorescence. Proc Natl Acad Sci USA 83:8361-8365, 1986. 10 Khalfan H, Abuknesha R, Rand-Weaver M, Price RG, Robinson D: Aminomethyl coumarin acetic acid: A new fluorescent labelling agent for proteins. Histochem J 18:497-499, 1986. 11 Kievits T , Devilee P, Wiegant J , Wapenaar MC, Cornelisse C J , van Omrnen GJB, Pearson PL: Direct non-radioactive in situ hybridization of somatic cell hybrid DNA to human lymphocyte chromosomes. Cytometry (submitted). 12. Landcgent J E , Jansen in de Wall N, Baan RA, Hoeijmakers J H J , van der Ploeg M: 2-acetylaminofluorene-modified probes for the indirect hybridocytochemical detection of specific nucleic acid sequences. Exp Cell Res 153:61-72, 1984. 13. Langer FR, Waldrop AA, Ward DC: Enzymatic synthesis of biotin labeled polynucleotides: Novel nucleic acid affinity probes. Proc Natl Acad Sci USA 78:6633-6637, 1981. 14. Nederlof PM, Robinson D, Abuknesha R, Wiegant J , Hopman AHN, Tanke H J , Raap AK: Three-color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences. Cytometry 10:20-27, 1989.
MUIJTIPLE E’LUORES(’ENCE IN SITIJ HYBRIDIZATION 15. Nederlof PM, van der Flier S, Raap AK, van der Ploeg M, Kornips F, Geraedts JPM: Detection of chromosome aberrations in interphase tumor nuclei by non-radioactive in situ hybridization. Cancer Genet Cytogenet 42:87-98, 1989. 16. Pinkel D, Landegent J, Collins C, Fucsoe J , Segraves R, Lucas J, Gray J: Fluorescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc Natl Acad Sci USA 85:9138-9142, 1988. 17. Sverdlov ED, Monastyrskaya GS, Guskova LI, Levitan TL, Sheichenko VI: Modification of cytidine residues with a bisulfite-
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o-methylhydroxylamine mixture. Biochim Biophys Acta 340: 153, 1974. 18. Waye JS, Willard HF: Molecular analysis of a deletion polymorphism in alpha satellite of human chromosome 17: Evidence for homologous unequal crossing-over and subsequent fixation. Nucleic Acids Res 145915, 1986. 19. Waye JS, England SB, Willard HF: Genomic organization of alpha satellite DNA on human chromosome 7: Evidence for two distinct alphoid domains on a single chromosome. Mol Cell Biol 7:349-356, 1987.
Cytometry 11:126-131 (1990)
Multiple Fluorescence In Situ Hybridization P.M. Nederlof, S. van der Flier, J. Wiegant, A.K. Raap, H.J. Tanke, J.S. Ploem, and M. van der Ploeg Sylvius Laboratories, Department of Cytochemistry and Cytometry, University of Leiden, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands Received for publication May 23, 1989; accepted J u n e 17, 1989
A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets
Chromosome banding techniques are widely applied for the identification of numerical and/or structural chromosome aberrations in tumor and prenatal diagnosis. The interpretation of the banding patterns requires skilled technicians; it is often complicated by imperfect banding, chromosome condensation, and limited numbers of metaphases; and i t is extremely difficult in case of highly aneuploid tumors with extensive structural changes. An alternative method for the detection of numerical chromosome aberrations is the in situ hybridization technique with chromosome specific repetitive probes, which also has the advantage that interphase nuclei can be analysed (3,5,8,15). One has to keep in mind that chromosome specific satellite probes only give information about the centromeric region of the chromosome, and therefore do not allow detection of (structural) chromosome aberrations which involve other parts of that chromosome. Recently, however, probes have been developed from chromosome specific libraries which can be used for overall staining of specific individual chromosomes by using competitor total genomic DNA to exclude dispersed repetitive sequences from participating in the in situ hybridization (2,161. As a n alternative to this type of probe “cocktail,” DNA from somatic cell hybrids containing specific chromosomes (or fragments) can be used (11).The combination of specific integral cytochemical chromosome staining procedure with chromosome specific satellite probes allows, in principle, detection of considerably more numerical and structural aberrations (translocations).
simultaneously is important for prenatal diagnosis and the detection of numerical andlor structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique. Key terms: Chromosomal aberrations, prenatal diagnosis, oncology, fluorescence ratio imaging
The possibility of detecting multiple sequences simultaneously is very important, since i t allows analysis of nuclei for different aberrations a t the same time. The number of simultaneously detectable target sequences by fluorescence in situ hybridization is determined by the number of specific probes, different labeling techniques, independent (immunological) detection methods, and microscopically detectable labels. With the large number of probe haptenization procedures available, it appears that currently the maximum number of simultaneously detectable probes is limited by the number of immunofluorophores available. Although in fluorescence microscopy and evaluation by the naked human eye the number of spectrally separated fluorochromes is practically limited to three colors (blue (AMCA); green (FITC); red (TRITC, Texas Red)), we show in this paper that the number of simultaneously detectable probes can be increased to more than three. This is achieved by combining double haptenized probes and three fluorochromes in such a way that in principle up to seven targets can be detected (Table 1). Preliminary results, using fluorescence digital image microscopy (FDIM) (l),indicate that the fluorescence intensity ratios of double haptenized probes
Address reprint requests to Dr. P.M. Nederlof, Sylvius Laboratories, Department of Cytochemistry, University of Leiden 2333 AL Leiden. The Netherlands.
are fairly constant. Thus by using probes with different hapten ratios and fluorescence ratio imaging, the number of simultaneously detectable probes can be increased even further. The benefit of multiple in situ hybridization for cytogenetic analysis lies primarily in the fact that automated and more objective identification of chromosome (segments) is feasible. Furthermore, multiple hybridizations with fluorescence ratio imaging in combination with high quality image representation using artificial color may form the basis of a n in situ hybridization chromosome banding procedure.
MATERIALS AND METHODS Preparation of the Microscopical Slides Metaphase preparations were obtained from phytohaemagglutinin stimulated human peripheral blood lymphocytes from a normal male (46, XY), which were treated for 1 h with Colcemid (0.5 pgiml). After centrifugation cells were resuspended in a prewarmed (37°C) swelling buffer containing 50 mM KC1, 10 mM MgSO,, 3 mM DTT, and 5 mM K2HP04 pH 8.0. After incubation for 30 min a t 37°C cells were fixed with four changes of methanoliacetic acid (3:l viv). Cell suspensions were stored a t -20°C. Cells were centrifuged on cleaned (ethanoliether, 1/ 1) slides, allowed to air dry (overnight), washed with phosphate-buffered saline (PBS: 0.15 m NaC1, 10 mM Na phosphate, pH 7.21, and gradually dehydrated with ethanol. Before use, slides were treated with 100 pgiml RNase A in 2 x SSC (0.3M NaC1,30 mM Na citrate, pH 7.2) under a coverslip for 60 min at 37"C, with proteinase K (0.1 ygiml in 20 mM Tris-HC1, 2 mM CaCl,, pH 7.4), for 7.5 min a t 37°C and were post-fixed with 4% (para)formaldehyde (in PBS, 50 mM MgC12)for 10 min, dehydrated, and kept a t room temperature until used.
DNA Probes and Labeling Human alphoid or simple satellite probes specific for chromosomes #1 (pUC1.77) (4),#7 (pa7tl) (19), #15 (D15Z1) (7), and #17 (p17H8) (18) were labeled with biotin (bio) according to Langer et al. (13),with amino acetylf luorene (AAF) according to Landegent et al. (121, or with the Chemiprobe kit (CP) (17) (FMC Bioproducts, generous gift of Amstelstad BV, Zwanenburg, NL) according to the manufacturer's instructions. In case of double haptenization with AAF and biotin, the (non-sonified) probes were first labeled with AAF, precipitated, and dissolved in a nick-translation buffer for labeling with bio-11-dUTP. For the combination Chemiprobeibiotin, probes were first labeled with CP.
Table 1 Scheme for Multiple Hybridization Combining Biotin, AAF, and Chemiprobe in Single-, Double-, and Triple-Probe Labeling
Label bio AAF CP biolAAF bioiCP AAFICP bioiAAFiCP
Red TRITC
Color signal Green FITC
+~
+ ++
Blue AMCA ~
+ ~
-
+ + +
-
+ + + +
In Situ Hybridization For each hybridization the labeled probe(s) was mixed in a hybridization buffer containing 60% deionized formamide, 2 x SSC, carrier DNA (50 times excess of salmon sperm DNA and yeast RNA). Per hybridization, 7 p1 of probe mixture is used (containing 20 ng for the probes: p#7-bio, p#15-AAF, p#l7--Cp, and 10 ng for p#l-AAFibio). The probes and target were denatured together under a coverslip (18x 18 mm) a t 80°C for 5 rnin in a n incubator. Hybridization was performed in a moist chamber for 16 h a t 37°C. Slides were then washed a t room temperature two times 5 min with 60% formamide, 2 x SSC, and two times 10 min with 2 x SSC. Detection After a wash with PBSiTween-20 for 5 min, slides were incubated a t 37°C for 30 min with 100 ~1 PBSi Tween-20 with 5% normal goat serum under a 24 x 50 mm coverslip. All immunological incubations were performed in PBS containing 0.05% Tween-20, 5% normal goat serum, and the appropriate dilution of antisera. Incubations were for 45 min a t 37°C in a moist chamber. After each incubation the slides were washed three times with PBS/O.O5%,Tween-20. For the detection of the double labeled probe ( p # l AAFibio) in the first layer a mouse anti-AAF monoclonal antibody (diluted 1500, kindly provided by Dr. R.A. Baan, TNO, Medical Biological Laboratories, Rijswijk) in combination with TRITC conjugated avidin (1:4000, Zymed, USA) was used. The second step contained FITC conjugated rabbit anti-mouse IgG, (1:800, Vector, USA). For the quadruple hybridization, the first layer consisted of a mouse monoclonal anti-Chemiprobe (from the Chemiprobe-kit) (1:800), a rabbit anti-AAF polyclonal antiserum (1500, R.A. Baan), and TRITC conjugated avidin (1:4,000).The second layer contained a n AMCA conjugated goat anti-mouse (1:200, Sigma, USA), prepared a s described by Khalfan e t al. (10) USing N-hydroxysuccimide-AMCA (Biocarb, Lund, Sweden) and a FITC conjugated goat anti-rabbit antibody (1:800, Sigma, USA).
MU1,TIPLE FLITOHESC'ENCE IN SITIT HYKI:IDIZATION
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FIG 3. Quadruple hybridization p#l-bioiAAF iTRITC/FITC), p#7-bio (TRITC), p#15-AAF iFITC), p#17-CP (AMCA),on a n interphase nucleus lymphocyte.
Microscopy
photomicrographs were taken with a Leitz ~ i microscope with a Vario-orthomat, equipped for FITC, TRITC, and AMCA fluorescence (14) using a 3~ color slide film. E~~~~~~~times were about 1-2 min for FITC, 30 s for AMCA, and 90 s for TRITC.
RESULTS AND DISCUSSION The different haptens used for the labeling of DNA probes bind to different nucleotides; it is possible, therefore, to double label one probe with two (or more) different haptens, which can be visualised in different colors. Biotin labeling was performed by nick-translation with bio-11-dUTP (the biotin is coupled to the C5 position of the uridine base); 2-acetyl aminofluorene
~ _ _ _ _
F!G 1. Single hybridization with a double labeled probe p#l-bio/ AAF (FITCITRITC) on lymphocyte metaphase chromosomes, DAPI counterstained. A. Double exposure with blue excitation (FITC) and UV excitation (DAPI). B: Double exposure with green excitation (TRITC) and UV excitation. FIG 2 . Triple hybridization p#l-bio/CP (FITC/TRITC), p#7-bio (FITC), p#17-CP (TRITC), on metaphase chromosomes, DAPI counterstained. A Double exposure with blue and UV excitation for, respectively, FITC and DAPI. B: Double exposure with green and UV excitation for TRITC and DAPI.
(AAF) is bound covalently to the C8 position of guanosine and the~ Chemiprobe kit labels the C6 ~ residues; l ~ Position of cytosine residues with a sulfonate group. Although modification with biotin and AAF alters the stability of the hybrid formation since it affects the base pairing, the double labeled probes retained their chromosome specificity under the hybridization conditions applied (60% formamide, 2 x SSC, 37°C). For double labeling with AAF and biotin best results were obtained when the AAF labeling was performed prior to the biotin labeling. The nick-translation reaction time was then reduced to 1h incubation (normally 2 h). Also double labeled probes combining AAF and Hg, or CP and biotin, were used, giving similar results (not shown). Instead of a double labeled probe, i t is also possible to use a mixture of two different single labeled probes (results not shown). In Figure 1 the results of a hybridization of a double labeled probe for chromosome #1 with AAF and biotin are given. On the metaphase chromosomes the AAF hapten is visualised in red (TRITC) and the biotin label in green (FITC). The FITC signal is very intense, and also some minor binding sites are visible. The fact that the green and red fluorescence signal are not exactly on the same position is caused by the need for changing filter blocks for FITC and TRITC, thereby slightly changing the orientation of the dichroic mirror. The availability of new filter blocks consisting of one excitation filter and dichroic mirror which allow simultaneous detection of FITC and Texas Red will eliminate this phenomenon.
By applying these double labeled probes in combination with only two single labeled probes it is possible to perform tripie hybridizations using only two fluorochromes (FITC: green, and TRlTC: red); this has the advantage that DAPI (blue) can be used a s total DNA counterstain. Probe p#l-biolCP was combined with p#7-bio and p# 17-CP in a triple hybridization experiment on human lymphocytes (Fig. a), showing chromosome #1 in both green and red, chromosome #7 in green, and chromosome #17 in red. Such a double labeled probe can also be used in a quadruple-hybridization, when used in combination with three single labeled probes. For the simultaneous detection of four targets in lymphocyte nuclei four different probes were used, the double labeled p#l-AAFI bio probe and three single labeled probes: p#7-bio, p#15-AAF and p#17-CP. In Figure 3 the results of this fourfold hybridization are shown: chromosome #1 is visible in both green and red, chromosome #7 in red, chromosome #15 in green, and finally chromosome #17 in blue. The number of different probes used in one in situ hybridization can in principle already be extended from four to seven by using three single labeled probes, three double labeled probes, and one triple labeled probe. Introduction of infra-red emitting cyanin dyes ( 6 ) can increase the multiplicity of in situ hybridizations even further. Visual interpretation will be complex, however, and impossible for the infra-red dyes. Therefore, digitizing of fluorescent images and visualization with pseudo-colors will be required. Fluorescence digital imaging microscopy using sensitive (charge-coupled device) cameras is ideally suited for this purpose (I).Preliminary measurements, using digital fluorescence imaging microscopy with an intensified video camera (Hamamatsu VIM system, C1966-20, Hersching, West Germany), so far show that the intranuclear intensities of in situ hybridization signals obtained with chromosome specific satellite probes are fairly constant. The internuclear variation is however considerable, probably due to differences in hybridization efficiencies. Internal standards and therefore, additional hybridizations will be necessary to correct for these internuclear differences. Furthermore, measurement of the FITC and TRITC spot intensities of double labeled (chromosome specific satellite) probes revealed that the FITCiTRITC intensity ratio is fairly constant (S.D. < 10%). For fast automated screening for chromosomal aberrations for prenatal diagnosis, the three autosomal chromosomes #13, #18, and #21 and the sex chromosomes are clinically the most important; therefore, the possibility to perform a fivefold hybridization is of great interest. When the hapten ratio on the probe can be controlled, measurement of the FITCITRITC ratio may allow identification of different probes on basis of their difference in hapten ratios. This will increase the number of detectable probes even more by using only three
colors (comparable to the use of two-color immunofluorescence in flow cytometry; ref. 9). This would create the possibility to detect in a n automated fashion multiple chromosomes by fluorescence ratio imaging, and may also be the basis for a n in situ hybridization banding technique using differently (double) labeled probe cocktails from chromosome (segment) specific libraries.
ACKNOWLEDGMENTS The authors thank Hamamatsu Photonics Europa GMBH (Herrsching, West Germany) for placing at our disposal the Hamamatsu VIM system.
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