TECHNICAL NOTE |
Correspondence to: Ernst J.M. Speel, Dept. of Molecular Cell Biology & Genetics, University Maastricht, PO Box 616, 6200 MD Maastricht, The Netherlands.
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Summary |
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We describe the simultaneous localization of DNA sequences in cell and chromosome preparations by means of differently fluorochrome-labeled (AMCA, FITC, TRITC) tyramides using the catalyzed reporter deposition (CARD) procedure. For this purpose, repeated as well as single-copy DNA probes were labeled with biotin, digoxigenin, and FITC, hybridized, and visualized with three different cytochemical detection systems based on horseradish peroxidase conjugates. These were sequentially applied to interphase nuclei and metaphase chromosomes at low concentrations to prevent crossreaction and nonspecific background. In situ localized peroxidase activity was visualized by the deposition of fluorochrome-labeled tyramide molecules. To allow specific deposition of a second and a third tyramide conjugate for multiple-target fluorescence in situ hybridization (FISH), remaining peroxidase activity was always completely inactivated by a mild acid treatment before application of the next peroxidase conjugate. The CARD reactions were optimized for maximal signal-to-noise ratio and discrete localization by tuning reaction time, H2O2, and tyramide concentrations. For both repeated and single-copy DNA targets, high FISH signal intensities were obtained, providing improvement of sensitivity over conventional indirect detection systems. In addition, the fluorescence CARD detection system proved to be highly efficient and easy to implement in multiple-labeling studies, such as reported here for FISH. (J Histochem Cytochem 45:1439-1446, 1997)
Key Words: FISH, tyramide, horseradish peroxidase, enzyme cytochemistry, DNA probes, interphase nuclei, metaphase spreads
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Introduction |
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During the past decade, fluorescence in situ hybridization (FISH) techniques have been shown to be important tools in a variety of molecular genetic and cell biological approaches (for reviews see
The latter method, introduced by
Here we describe the use of AMCA-, FITC-, and TRITC-labeled tyramides (TSA direct; NEN Life Science Products, Boston, MA) in sensitive multiple-target FISH procedures, enabling the detection of both repeated and single-copy DNA sequences in interphase cells and metaphase spreads (see also
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Materials and Methods |
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Cell Samples
Metaphase spreads were prepared from human peripheral blood lymphocytes by phytohemagglutinin stimulation, hypotonic spreading, and fixation in methanol:acetic acid (3:1, v/v) according to standard procedures. A single-cell suspension of the human transitional cell carcinoma line T24 (DNA index 1.6; trisomic for the centromeres of chromosome 1 and 7; tetrasomic for the centromere of chromosome 17 and the telomere of chromosome 1p) was made by trypsinization of cultured cells, followed by harvesting and fixation in 70% ethanol as described by
Pretreatment of Cell Samples
Pretreatment of metaphase spreads with RNAse A and pepsin, followed by postfixation in 1% formaldehyde in PBS (0.15 M NaCl, 10 mM sodium phosphate, pH 7.2) has been described previously (
Removal of the cytoplasm of T24 cells to improve DNA probe and conjugate penetration was performed as described earlier (
DNA Probes and Probe Labeling
DNA probes specific for the (sub)centromeric regions of human chromosomes 1 (pUC 1.77), 7 (p7t1), 8 (D8Z2), 11 (pLC11A), 17 (p17H8), the telomeric region of chromosome 1 (p1-79), the 5S rDNA repeat on chromosome 1q42-43 (pH5SB), chromosome 11p15 and 11q23 (40-KB cosmids cCII11-310 and cCII11-314), and chromosome 8q24 (five plasmid clones representing 15.8 KB of the thyroglobulin gene) have been described earlier (
FISH Procedures
The labeled (sub)centromere, telomere, cosmid, and plasmid probes were hybridized in single- and multiple-target FISH procedures on metaphase spreads, T24, and TCC 9 cells as described earlier (
Cytochemical Probe Detection
After these washing steps, the slides were preincubated with 4 x SSC, pH 7.0, containing 5% nonfat dry milk (Buffer B) for 10 min at 37C to reduce background staining, followed by dipping in Buffer A. For all the detection procedures, the avidin conjugates were diluted in Buffer B and all the antibody conjugates were diluted in PBS containing 0.05% Tween 20 (Buffer C) and 2% normal goat serum (NGS). After each incubation step of 30 min at 37C, the slides were rinsed twice in Buffer A (avidin conjugates) or Buffer C (antibody conjugates) for 5 min at RT.
Biotin-labeled probes were detected with horseradish peroxidase-conjugated avidin (AvPO, 1:200; DAKO, Glostrup, Denmark) and, optionally, with subsequent layers of biotinylated goat anti-avidin (BioGAA, 1:200; Vector, Brunschwig Chemie, Amsterdam, The Netherlands) and AvPO, or with mouse anti-biotin (MABio, 1:400; DAKO) and horseradish peroxidase-conjugated goat anti-mouse IgG (GAMPO, 1:200; DAKO). For visualization with the alkaline phosphatase-Fast Red reaction, probes were detected with alkaline phosphatase-conjugated avidin (AvAPase, 1:50; DAKO). Digoxigenin-labeled probes were detected with horseradish peroxidase-conjugated sheep anti-digoxigenin Fab fragments (ShADigPO, 1:200; Boehringer), used only for the detection of the (sub)centromeric probes, or with mouse anti-digoxin (MADig, 1:20000; Sigma) and GAMPO. FITC-labeled probes were detected with horseradish peroxidase-conjugated anti-FITC (AFITCPO, 1:2000; NEN Life Science), used only for the detection of the (sub)centromeric probes, or with rabbit anti-FITC (RAFITC, 1:2000; DAKO) and horseradish peroxidase-conjugated swine anti-rabbit IgG (SWARPO, 1:200; DAKO).
Multiple-target detection was carried out essentially as described earlier (
The largest DNA targets were always detected last in sequence, which occasionally involved a change in the sequence of probe detection. In all cases, control experiments with fluorochrome-conjugated avidin or fluorochrome-conjugated antibody molecules were performed to check the specificity of the observed peroxidase-tyramide staining patterns. In some cases the digoxigenin-labeled probes were detected by subsequent incubations with TRITC-conjugated sheep anti-digoxigenin Fab fragments (ShADigTRITC, 1:100; Boehringer)/FITC-conjugated avidin (AvFITC, 1:500; Vector, Burlingame, CA), BioGAA and AvFITC, or by a sequential application of AMCA-conjugated avidin (AvAMCA, 1:100; Vector), BioGAA, AvAMCA, MADig (1:2000), and TRITC-conjugated rabbit anti-mouse IgG (RAMTRITC, 1:100; DAKO).
Enzyme Cytochemistry
Peroxidase Cytochemistry with Fluorochrome-labeled Tyramides (CARD Amplification).
After cytochemical detection of the probes, peroxidase detection was performed by addition of different dilutions of hydroxycoumarin (AMCA)-, carboxyfluorescein (FITC)-, or rhodamine (TRITC)-labeled tyramide stock solutions (1 mg/ml; kindly provided by NEN Life Science) in 100% ethanol (AMCA-tyramide) or PBS (FITC- and TRITC-tyramides) to 1 x Amplification Diluent (NEN Life Science) or PBS containing 0.1 M imidazole, pH 7.6, and 0.001% H2O2, and application of this reaction mixture to the slides (50 µl under a coverslip) for 5-15 min at 37C. The slides were washed two times for 5 min with Buffer C. Currently, the fluorochrome-labeled tyramides are commercially available in TSA (tyramide signal amplification) kits from NEN Life Science.
Alkaline Phosphatase Cytochemistry with Fast Red TR.
The alkaline phosphatase-Fast Red reaction was performed as described earlier (
Embedding and Microscopy
Slides were embedded in 90% glycerol/0.02 M Tris-HCl, pH 8.0, containing 2% 1,4-di-azobicyclo-(2,2,2,)-octane (DABCO; Sigma) and, optionally, 0.5 µg/ml 4',6-diamidino-2-phenyl indole (DAPI; Sigma), propidium iodide (PI; Sigma), or YOYO (Molecular Probes; Eugene, OR). Slides were analyzed using a Leica DM fluorescence microscope with appropriate filter sets for AMCA, FITC, and TRITC, coupled to a CCD camera and image processing system (MetaSystems; Heidelberg, Germany). Images were recorded using a x63 or x100 objective and corrected for background. It should be emphasized here that although a CCD imaging system was used, the FISH signals described in this report were clearly visible through the microscope.
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Results |
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A multiple-target FISH detection procedure was developed applying multiple enhancement steps with the CARD amplification system. For this purpose, combinations of differently fluorochrome-labeled tyramides (TSA direct) were tested in several cell systems, including interphase preparations of T24 and TCC9 tumor cells and metaphase spreads of normal human peripheral blood lymphocytes. The individual enzyme reactions had been optimized first in single-target FISH procedures before performance of the multiple-target FISH procedures.
Single-target FISH
Discrete in situ localization of different DNA targets using the CARD system as the final detection step could be achieved by optimizing conjugate dilutions, H2O2, and fluorochrome-labeled tyramide concentrations in the reaction medium, and incubation time (5-15 min). Optimal and accurate localization of hybridized (sub)centromeric probes was achieved by using the following:
Figure 1A-C show the results of the detection of a biotinylated chromosome 1 probe with AvPO and the three different CARD reactions in T24 cells. All cells display three copies of the chromosome 1 centromere, with accurately localized FISH signals of high signal intensities. The signal intensities were considerably higher than those obtained with conventional, indirect detection methods, enabling a two- to tenfold reduction of CCD recording times while still preserving a higher intensity of FISH signals in most cases over conventional generated spots (for comparison see Figure 1I-J and Figure 1L-M). TRITC-tyramide depositions provided the highest sensitivity. In comparison with the produced TRITC-tyramide signals (Figure 1C), similarly intense FISH signals could be generated by the alkaline phosphatase-Fast Red reaction, until now one of the few enzyme reactions producing an accurately localizing fluorescent precipitate. For this purpose, standard dilutions of detection conjugates were used and an enzyme reaction time of 5 min (Figure 1D).
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Distinct visualization of smaller DNA targets (1p36 repeat and 5S rDNA repeat, both several 100 KB, the 40-KB 11p15 and 11q23 cosmids, and the 15.8-KB thyroglobulin gene single-copy targets) by CARD detection was accomplished using standard probe concentrations of 5 ng/µl. Similar conditions were used as described for the repeated target probes, with the exception that two or three cytochemical detection layers need to be used as well as CARD reactions of 5-15 min. Application of only one detection layer in most cases led to inefficient and asynchronous amplification of the probe signals (data not shown). Results are illustrated in Figure 1, showing cosmid hybridization on chromosome 11q23 (Figure 1E), and, as part of multiple-target FISH experiments, thyroglobulin gene detection on chromosome 8q24 (Figure 1G), four copies of the 1p36 repeat in T24 cells (Figure 1H), two copies of the 11p15 cosmid sequence in TCC 9 cells (Figure 1J), and the 1p36 and 5S rDNA repeats on chromosome 1p36 and 1q42-43 (Figure 1M). On metaphase preparations, the low- and single-copy sequences could be resolved in most cases on both sister chromatids (Figure 1E and Figure 1M). The use of Alu sequence-containing probes, such as the two cosmids and the 5S rDNA probe, usually requires the addition of a 10- to 100-fold excess of competitor DNA (total human placenta or Cot1 DNA) to the hybridization mixture to block nonspecific hybridization and thus to obtain a maximal signal-to-noise ratio. For CARD detection of these probes, however, higher competitor concentrations were needed (up to 500-fold) to obtain optimal signal-to-noise ratios. This is most probably due to the fact that during CARD reactions nonspecific hybridization spots, barely detectable with conventional detection systems, are also amplified to a considerable extent, resulting in reduced signal-to-noise ratios after CARD detection at competitor concentrations (10- to 100-fold excess) usually added to the probes.
Double-target FISH
Double-target FISH was performed by hybridizing a biotinylated and a digoxigenin-labeled probe simultaneously on the same metaphase spread or interphase cell preparation and combining the TRITC-tyramide deposition with either the FITC- or AMCA-tyramide deposition reaction for detection. In this way, two copies of the centromeres for chromosomes 1 (FITC-tyramide) and 8 (TRITC-tyramide) could be visualized efficiently in the same metaphase spreads (Figure 1F), while the two chromosome 8 centromeric regions (FITC-tyramide) were detected together with the 15.8-KB thyroglobulin target sequence on chromosome 8q24 (TRITC-tyramide, Figure 1G). Furthermore, T24 and TCC 9 cells were used to detect an imbalance between the telomeric 1p36 region (four copies, TRITC-tyramide) and the centromere of chromosome 1 (three copies, FITC-tyramide) (Figure 1H), and an imbalance was also detected between chromosome region 11p15 (two copies, TRITC-tyramide) and the chromosome 11 centromere (three copies, FITC-tyramide; Figure 1J). In the latter two cases, the smallest DNA target was always visualized first. Other combinations of fluorochrome-conjugated tyramides could also be used efficiently whenever needed, together with the appropriate DNA counterstain (blue DAPI, green YOYO, or red PI; data not shown). Inactivation of peroxidase rest activity in between the two tyramide deposition reactions was essential, because omission of this step led to repeated deposition of the second tyramide in the vicinity of the DNA target detected first. For this purpose, the slides were incubated with 0.01 N HCl for 10 min at RT immediately after the first peroxidase-tyramide reaction. Neither the final FISH results nor the cell morphology were affected by this procedure.
To determine the effect of the CARD amplification procedure on the number of finally observed FISH signals per nucleus, this procedure was compared with conventional detection on TCC 9 cells, hybridized with the chromosome 11 centromere and 11p15 cosmid probe (e.g., see Figure 1I-J). For this purpose, 100 intact, nonoverlapping cells were evaluated for both DNA probes. With conventional detection, the main 11c/11p15 ratios were 3/2 (76% of the cells) and 2/2 (12% of the cells), whereas after CARD detection 72% of the cells showed the 3/2 ratio and 14% a ratio of 2/2. The efficiency of DNA target detection is therefore not influenced by the use of CARD detection systems, whereas in the latter case spot counting was much easier due to the high intensity of the FISH signals.
Triple-target FISH
The AMCA-, FITC-, and TRITC-tyramide deposition reactions were combined in triple-target FISH procedures for simultaneous visualization of the centromeres of chromosomes 1 (three copies, FITC), 7 (three copies, TRITC) and 17 (four copies, AMCA) in T24 cells (no counterstaining applied; Figure 1K). As already described for the double-target FISH procedure, mild acid treatment was essential for inactivation of peroxidase rest activities after each CARD reaction. Another example shows the probes for 5S rDNA, the chromosome 1 centromere, and 1p36 on metaphase spreads, detected with AMCA-, FITC-, and TRITC-tyramides, respectively (Figure 1M). In this case, a high detection efficiency could also be achieved with much higher signal intensities than after conventional detection (Figure 1L).
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Discussion |
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In the present study we describe the further optimization and application of the new CARD amplification method for use in multiple-target FISH procedures. A combination of three DNA probes, haptenized with either biotin, digoxigenin, or FITC and hybridized simultaneously to interphase or metaphase preparations, could be cytochemically detected by sequential application of three unrelated, peroxidase-conjugated detection systems, each directly followed by the peroxidase-mediated deposition of blue AMCA-, green FITC-, or red TRITC-labeled tyramides (TSA direct). In this way, three different DNA sequences (repeated as well as single-copy targets) could be localized simultaneously with high sensitivity and efficiency for immediate analysis by fluorescence microscopy. The entire detection procedure, including peroxidase inactivation steps (see below), could be carried out within 3 hr, which was only slightly longer than the conventional detection procedure.
The CARD detection protocol using standard probe concentrations was optimized for maximal signal-to-noise ratio and discreteness of signal localization by adjusting the number of cytochemical detection layers, the dilution of the reagents, the H2O2, and the tyramide concentrations in the reaction medium, as well as the incubation time of the enzyme reaction. When DNA probes containing repetitive elements, such as Alu sequences, were used, a high excess of competitor DNA was a prerequisite to prevent a reduction of the signal-to-noise ratio caused by the CARD amplification reaction. This was also essential for using even more complex probes, such as Alu- or DOP-PCR-amplified sequences, and sometimes needed, in addition, decreasing probe concentrations to obtain acceptable signal-to-noise ratios (data not shown).
In general, detection conjugates could be diluted two- to tenfold further than for conventional detection. Concentrations of AMCA- and FITC-labeled tyramides were found to be optimal at a dilution of 1:250, whereas TRITC-tyramide proved to be the most sensitive, as has been reported similarly by
Although we have not yet quantified the FISH signal intensities after CARD amplification and compared the results with conventionally developed signals, in most cases CCD recordings of these images were two- to tenfold shorter than for conventional detection procedures. For example, the exposure times for Figure 1M were 2.6, 0.44, and 0.24 sec for AMCA, FITC, and TRITC CARD images, respectively, whereas they were 5.0, 1.92, and 3.8 sec for the conventional images in Figure 1L. This suggests an amplification factor of at least two- to tenfold with acceptable resolution of the CARD signals.
It is now generally assumed that intensification of FISH signals observed after CARD amplification results from the binding of peroxidase-generated tyramide radicals to tyrosine moieties present in the (fixed) cell preparations. It was to be expected that the use of cytochemical blocking steps, such as nonfat dry milk, increases the binding sites for tyramide radicals. However, experiments comparing different blocking reagents (nonfat dry milk, 5% normal goat serum, and 3% BSA) with no blocking on T24 cells resulted only in slightly different staining intensities, suggesting that application of blocking steps is not essential for optimal signal generation.
The utilization of multiple CARD reactions on one sample requires efficient peroxidase inactivation steps in between two subsequent enzyme reaction steps. The 0.01 N HCl inactivation procedure, which we also used for multiple-target in situ hybridization combined with brightfield microscopy (
We conclude that the fluorescence CARD detection of multiple DNA targets in situ can be applied efficiently and rapidly on different cell preparations, combining the high sensitivity of the CARD system with only minor loss of resolution. This method will be of great value to further increase the detection sensitivity of FISH on different biological materials. Moreover, it will facilitate the counting of FISH signals, which can be advantageous for evaluation of chromosomal aberrations in cytological and histological material.
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Acknowledgments |
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Supported by the Netherlands Organization for Scientific Research NWO, grant no. 900-534-102, and by the Dutch Cancer Foundation, grant no. IKL 92-07.
Received for publication January 17, 1997; accepted May 15, 1997.
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