TECHNICAL NOTE |
Correspondence to: Anton H.N. Hopman, Dept. of Molecular Cell Biology and Genetics, University Maastricht, PO Box 616, 6200 MD Maastricht, The Netherlands.
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Summary |
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A one-step procedure for the synthesis of different tyramide conjugates, which can be utilized in the catalyzed reporter deposition (CARD) amplification system, is described. Succinimidyl esters of biotin, digoxigenin, and of the fluorochromes fluorescein, rhodamine, aminomethylcoumarine acetic acid, and Cy3 were coupled to tyramine in dimethylformamide (DMF) adjusted to a pH of 7.08.0 with triethylamine (TEA). The coupling reaction can be performed within 2 hr and the reaction mixture can be applied without further purification steps. Furthermore, trinitrophenyl (TNP)-tyramide was prepared by adding 2,4,6,-trinitrobenzenesulfonic acid to tyramine dissolved in either MilliQ/DMF basified with TEA or in an NaHCO3 (pH 9.5) buffer. A subsequent precipitation of the TNPtyramide resulted in a high-yield isolation of this conjugate. The synthesized tyramide conjugates were applied successfully in single- and multiple-target in situ hybridization (ISH) procedures to detect both repetitive and single-copy DNA target sequences in cell preparations with high efficiency. The described approach provides an easy and fast method to prepare a variety of tyramide conjugates in bulk amounts at relatively low cost.
(J Histochem Cytochem 46:771777, 1998)
Key Words: (F)ISH, horseradish peroxidase, interphase cytogenetics, fluorescent labeling, enzyme cytochemistry, tyramide conjugates, DNA probes
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Introduction |
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After its introduction by Bobrow et al. in 1989, the Catalyzed Reporter Deposition (CARD) amplification procedure has been implemented successfully as a detection system in several molecular procedures, such as ELISA, Western blotting, immunocytochemistry, and in situ hybridization (ISH) (
Free primary amino groups can be quantitatively acylated by NHS esters in organic media within a few minutes and with minimal risk of hydrolysis of the active esters, leading to inefficient coupling or impure products (
The applicability of the newly synthesized tyramides was tested in single- and multiple-target ISH procedures visualizing both repetitive and single-copy target sequences in cell preparations with CARD amplification fluorescence or brightfield detection systems.
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Materials and Methods |
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Reagents for the Synthesis of Tyramide Conjugates
The N-hydroxysuccinimide esters of biotin [sulfosuccinimidyl-6-(biotinimide)hexanoate], abbreviated as BIO-NHS (MW 557), fluorescein [5-(and 6)-carboxyfluorescein] succinimidyl ester (FITC-NHS), abbreviated as FLU-NHS (MW 473), rhodamine [5-(and 6)-carboxytetramethylrhodamine] succinimidyl ester (TRITC-NHS), abbreviated as RHO-NHS (MW 528), and coumarin (succinimidyl-7-amino-4-methylcoumarin-3-acetic acid (AMCA-NHS; MW 330) were purchased from Pierce (Rockford, IL). The N-hydroxysuccinimide esters of digoxigenin (digoxigenin-3-0-succinyl--aminocaproic acid-N-hydroxysuccinimide ester; DIG-NHS, MW 659) and Cy3 (succinimidyl ester; Cy3-NHS, MW 766; one cap contains 200 mg) were purchased from BoehringerMannheim (Mannheim, Germany) and Amersham Life Science, (Arlington Heights, IL), respectively. Tyramine-HCl (MW 173) was purchased from Sigma Chemical (St Louis, MO), and dimethylformamide (DMF; water-free) and triethylamine (TEA; 7.2 M) were purchased from Pierce.
Tyramide Synthesis in Organic Media
Table 1 summarizes the different amounts of active esters (X-NHS), tyramine, and TEA to be combined in a one-step synthesis of tyramide conjugates. The NHS esters are prone to hydrolysis and are light-sensitive. For this reason, the active esters were freshly dissolved only shortly before tyramide synthesis. All esters were dissolved in DMF at concentrations as indicated in Table 1 to obtain an active ester stock solution "A" (10 mg per ml DMF). The tyramine-HCl stock solution "B" was prepared by dissolving 10 mg/ml DMF (equivalent to 58 µmol) to which a 1.25-fold equimolar amount of TEA (10 µl of 7.2 M stock solution; 72.5 µmol) was added. For efficient acylation, the active esters (stock solution A) were added in 1.1 times equimolar amounts to the tyramine stock solution B and left at room temperature (RT) in the dark for 2 hr. The synthesized tyramide conjugates were diluted with ethanol to obtain stock solution "C" of 1 mg/ml. The tyramide stock solutions could be safely stored for at least 8 months at 4C without any reduction in reactivity.
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Our synthesized BIOtyr was compared to the BLAST kit obtained from Du Pont NEN (Boston, MA) and the Catalyzed Signal Amplification (CSA) System from DAKO (Glostrup, Denmark). Our fluorochrome tyramide conjugates were compared with the TSA-Direct system from NEN Life Science Products (Renaissance TSA amplification kit).
TNPTyramide Synthesis in Aqueous Media
TNPtyr was prepared by two different protocols: (a) by mixing equimolar amounts of 2,4,6-trinitrobenzenesulfonic acid (TNBS) (MW 690, 5% solution in water; Pierce) and tyramine with an excess of TEA to obtain a pH of 9.5. The reaction was performed for 2 hr at RT; and (b) by mixing equimolar amounts of TNBS (20 mg/400 ml) and tyramine dissolved in 0.3 M NaHCO3 buffer, pH 9.5. This reaction was performed for 1 hr in the dark at RT in a 10-ml plastic tube, with mixing on a vortex every 15 min. Even after the first 15 min a precipitate of TNPtyr was formed. To complete the reaction the mixture was left at RT for another 1 hr. The precipitate was pelleted, and the supernatant was removed and washed subsequently with the carbonate buffer and MilliQ. The pellet was dried in a vacuum excitatory and dissolved in 0.2 ml DMF to which 0.8 ml ethanol was added.
Cell Processing
A 70% ethanol suspension of the human transitional cell carcinoma line T24 (
DNA Probes and Labeling Procedures
The probes for chromosome 1q12 (pUC 1.77), 1p36 (p179), 1q4243 (pH5SB), and 4p16 (c5.5) have been described by Cooke and Hindley (1979),
ISH Procedure
The DNA probes described above were used at a concentration of 0.4 ng/µg (pUC 1.77), 1 ng/µg (p179), or 5 ng/µg (pH5SB and c5.5) and hybridized in different combinations in a hybridization buffer containing 50% formamide, 2 x SSC (0.3 M NaCl, 30 mM Na-citrate) pH 5.0, 10% dextran sulfate, 0.2 mg/ml herring sperm DNA as carrier DNA, and 0.2 mg/ml yeast tRNA as carrier RNA. An excess of total human placenta DNA was added as competitor DNA when c5.5 (100 x excess competitor) or pH5SB (250 x excess competitor) was included. Ten µg hybridization buffer was added to each slide under a coverslip (20 x 20 mm). Denaturation was performed on the bottom of a metal box in a water bath at 70C for 3 min and hybridization was performed overnight at 37C. The slides were washed twice for 5 min at 42C with 2 x SSC containing 0.05% Tween 20, followed by two 5-min washes at 60C 0.1 x SSC and a 5-min wash with 4 x SSC, pH 7.0, containing 0.05% Tween 20 (Buffer A) at RT.
Cytochemical Detection Procedures
To reduce background staining in the cytochemical detection procedures, the slides were preincubated for 10 min at 37C with 4 x SSC, pH 7.0, containing 5% nonfat dry milk (Buffer B), followed by dipping in Buffer A (see above). 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 2030 min at 37C, the slides were rinsed twice in Buffer A in the case of avidin conjugates (ABC system) or Buffer C in the case of antibody conjugates.
Single-target ISH
The biotinylated pUC 1.77 probe was detected by peroxidase (PO)-conjugated avidin (Av-PO, 1:50 dilution; DAKO), followed by CARD amplification as previously described (
The tyramide conjugates were detected as follows: TNPtyr was detected with rabbit anti-DNP (RADNP 1:100; DAKO) and FITC-labeled swine anti-rabbit IgG (SWARFITC 1:80; DAKO); DIGtyr with FITC-labeled rabbit anti-DIG (RADIGFITC 1:100; Boehringer); BIOtyr with FITC-labeled avidin (AvFITC 1:500; Vector, Brunschwig Chemie, Amsterdam, The Netherlands). The digoxigenin-labeled c5.5 probe was visualized by peroxidase (PO)-conjugated sheep anti-DIG Fab fragments (SHADIG-PO 1:100; Boehringer), followed by CARD amplification with DIGtyr (see above), SHADIGPO, and the PODAB reaction (see above).
Multiple-target ISH
For multiple-target ISH with fluorochrome-labeled tyramides, CARD detection of different probes was performed sequentially, with a 0.01 M HCl treatment for 10 min at RT between the individual detection steps to inactivate PO activity (
PODAB and APaseFast Red reactions
PODAB Reaction (Brown Precipitate;
APaseFast Red Reaction (Red Precipitate;
Microscopy
Microphotographs were made on a Leica DM RBE microscope equipped with the Metasystem Image Pro System (black-and-white CCD camera; Sandhausen, Germany). Images were captured using the ISIS program for FISH and for brightfield ISH additional green, red, and blue filters were applied. Images were processed in Adobe Photoshop 3.0 at a resolution of 120 pixels/inch.
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Results |
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Tyramide Conjugate Synthesis
Table 1 summarizes the synthesis of seven different tyramide conjugates. For this purpose, a 1.1-fold molar excess of the active hapten- (BIO, DIG) or fluorochrome- (RHO, FLU, Cy3, AMCA) succinimidyl esters, freshly dissolved in DMF, was added to the tyramine. The tyramine was also dissolved in DMF adjusted with TEA to obtain a pH of about 7.08.0. After a 2-hr reaction, the synthesized tyramide conjugates were diluted to a final concentration of 1 mg/ml by adding ethanol without purification. These stock solutions were further diluted to working concentrations for ISH probe detection (see below), as indicated in Table 2.
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The TNPtyramide was synthesized by mixing the aqueous TNBS solution with tyramineTEA (see above) or in 0.3 M NaHCO3 buffer, pH 9.5. Because TNBS is not prone to hydrolysis in water, the coupling reaction can be performed with an excess of base in both organic and aqueous reaction media. During the reaction of TNBS with tyramine in 0.3 M NaHCO3 buffer, the produced TNPtyramide precipitated spontaneously, because these molecules are lipophilic and only slightly water-soluble. This precipitation reaction therefore allows the easy isolation of a pure reaction product while the unreacted tyramine and TNBS remain in solution. In this way, large amounts of this conjugate can be synthesized and isolated. The TNPtyramide is dissolved in DMF and diluted with ethanol.
Application and Sensitivity of Tyramide Conjugates in Fluorescence ISH
All synthesized tyramides were tested in single-target ISH experiments on T24 cells, using the biotinylated probe for 1q12 and one detection layer of AvPO. The fluorochrome-labeled tyramides (Cy3, FLU, RHO, and AMCA) were deposited in CARD amplification reactions for 5 min at 37C and could be visualized directly in the fluorescence microscope. T24 cells displayed three ISH signals per nucleus in all cases, as shown in Figure 1a for Cy3tyramide. Depositions of the hapten-labeled tyramides (BIO, DIG, and TNP) required additional incubations with fluorescent detection conjugates to visualize them in the fluorescence microscope. For example, Figure 1b and Figure 1c show the detection of chromosome 1q12 with TNPtyramide/RADNP/SWARFITC and DIGtyramide/SHADIG-FITC, respectively. Table 2 summarizes the working concentrations of the tyramide conjugates in the CARD amplification reactions for optimally localized fluorescent signals. All the tyramides could be applied at concentrations of 210 µM in a 510-min amplification reaction at 37C in PBSimidazole. The RHOtyramide appeared to be the most sensitive conjugate, as was to be expected on the basis of previously described observations (
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The applied tyramide conjugate concentrations were compared to the conditions recommended in the commercially available kits (BLAST, TSA Renaissance systems, or C.S.A. kits) and with the concentrations known from the literature (
The synthesized fluorochrome-conjugated tyramides were also tested in multiple-target FISH procedures. Figure 1d shows a double-target FISH using AMCAtyramide and Cy3tyramide. The chromosomal targets 1q12 and 1p36 were subsequently detected, with SADIGPO/Cy3tyramide and AvPO/AMCAtyramide, resulting in discrete CARD signals. For this purpose, the PO activity remaining after the first CARD reaction step was inactivated by a mild acid incubation step before application of the next CARD detection step. In this way a triple-target fluorescence ISH experiment with CARD detection could also be performed. Figure 1e shows the localization of the 1q4243, 1q12, and 1p36 targets on human chromosome 1 with AMCAtyramide, RHOtyramide, and FLUtyramide, respectively. The chromosomes were not counterstained to avoid color overlap of the general DNA stains (DAPI or PI) with one of the CARD signals.
CARD Amplification in Brightfield ISH
The haptentyramides could also be applied to localize both repetitive and single-copy gene DNA sequences in brightfield ISH. As an example, Figure 1f shows the detection of the digoxigenin-labeled c5.5 cosmid probe with SHADIGPO, DIGtyramide, SHADIGPO and the PODAB reaction. In the T24 interphase nuclei, both three and four ISH signals per nucleus could be visualized, as was expected from earlier unpublished results using fluorescence ISH (78% trisomy, 8% tetrasomy). Double-target brightfield ISH was carried out to detect 1q12 with APaseFast Red, after which the 1p36 target could be detected using the BIOtyramide. The latter target was detected in PODAB (Figure 1g).
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Discussion |
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The one-step synthesis of tyramide conjugates in DMF (adjusted to pH 7.08.0 with TEA) resulted in products that could be successfully applied in the CARD amplification step for ISH. The hydrolysis of the active fluorochrome or hapten NHS esters was circumvented by performing the reaction in water-free medium and by adding TEA to deprotonate the amino group of tyramine-HCl.
The synthesized tyramide conjugates were applied in model systems to test the sensitivity of immunocytochemical amplification systems. All synthesized products could be applied within the same concentration range, which indicates the efficiency of the synthesis, even though the purity of the products was not explicitly tested. Furthermore, for the known tyramides, these concentrations are comparable to the range of lowest concentrations reported in the literature (Table 2). The applicability and sensitivity of the haptenized tyramides in brightfield ISH are illustrated in Figure 1f and Figure 1g. The intense signals for the cosmid probes illustrate the sensitivity and are comparable with the results obtained by
In conclusion, our approach for the synthesis of the tyramide conjugates is an easy method to prepare a variety of new substrates for peroxidase cytochemistry because many active esters of fluorochromes or other haptens are commerically available. The protocol minimizes problems arising from dissolving of these active esters and facilitates the coupling reaction.
Furthermore, the protocol is suitable for bulk production of tyramide conjugates. In particular, TNPtyramide can be synthesized in gram quantities with high purity, opening the possibility of using this tyramide in a routine setting on a daily basis. In diagnostic pathology as well as other immunochemical studies, this could mean a significant reduction in the costs for primary antibodies, because these reagents can be diluted several more magnitudes.
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Footnotes |
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1 Present address: Dept. of Pathology, University Hospital Zurich, Switzerland.
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Acknowledgments |
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Supported in part by the Dutch Cancer Foundation, grant no. IKL 92-07.
Received for publication August 8, 1997; accepted December 11, 1997.
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