Journal of Histochemistry & Cytochemistry

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Phosphorescent Platinum/Palladium Coproporphyrins for Time-resolved Luminescence Microscopy Richard R. de Haas, Rob P.M. van Gijlswijk, Erik B. van der Tol, Jacky Veuskens, Hilde E. van Gijssel, Roeline B. Tijdens, Jan Bonnet, Nico P. Verwoerd and Hans J. Tanke J Histochem Cytochem 1999 47: 183 DOI: 10.1177/002215549904700207 The online version of this article can be found at: http://jhc.sagepub.com/content/47/2/183

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Volume 47(2): 183–196, 1999 The Journal of Histochemistry & Cytochemistry

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ARTICLE

Phosphorescent Platinum/Palladium Coproporphyrins for Time-resolved Luminescence Microscopy Richard R. de Haas, Rob P.M. van Gijlswijk, Erik B. van der Tol, Jacky Veuskens, Hilde E. van Gijssel, Roeline B. Tijdens, Jan Bonnet, Nico P. Verwoerd, and Hans J. Tanke Laboratory for Cytochemistry and Cytometry (RRDH,RPMVG,JB,NPV,HJT), Leiden University Medical Center, Leiden; Department of Organic Chemistry (EBVDT), University of Amsterdam, Amsterdam; Institute for Molecular Cell Biology (JV), University of Amsterdam; and Division of Toxicology, Leiden/Amsterdam Center for Drug Research (HEVG,RBT), Sylvius Laboratories, University of Leiden, Leiden, The Netherlands

Streptavidin and antibodies were labeled with phosphorescent platinum and palladium coproporphyrin. The optimal conjugates were selected on the basis of spectroscopic analysis (molar extinction coefficient, quantum yield, lifetime) and using ELISA assays to determine the retention of biological activity and immunospecificity. They were subsequently tested for the detection of prostate-specific antigen, glucagon, human androgen receptor, p53, and glutathione transferase in strongly autofluorescent tissues. Furthermore, platinum and palladium coproporphyrin-labeled dUTPs were synthesized for the enzymatic labeling of DNA probes. Porphyrin-labeled DNA probes and porphyrin-labeled streptavidin conjugates were evaluated for DNA in situ hybridization on metaphase spreads, using direct and indirect methods, respectively. The developed in situ detection technology is shown to be applicable not only in mammals but also in plants. A modularbased time-resolved microscope was constructed and used for the evaluation of porphyrinstained samples. The time-resolved module was found suitable for detection of antigens and DNA targets in an autofluorescent environment. Higher image contrasts were generally obtained in comparison with conventional detection systems (e.g., fourfold improvement in detection of glutathione transferase). (J Histochem Cytochem 47:183–196, 1999)

SUMMARY

Fluorescence has several features that make it attractive for application in biomedical science, such as specificity, sensitivity, and high temporal and spatial resolution. The introduction of new fluorescent dyes with improved photophysical properties, such as the cyanine, bodipy, and alexa dyes, in combination with the introduction of highly sensitive detection devices has made fluorescence microscopy an indispensable technique in molecular cell biology (Jovin and Arndt–Jovin 1989; Tanke et al. 1995). In addition to these improvements, the optimization of microscope stands and lens performance and the development of anti-fading reagents has further improved the detec-

Correspondence to: Richard R. de Haas, Lab. for Cytochemistry and Cytometry, Dept. of Molecular Cell Biology, Leiden Univ. Medical Center; Sylvius Laboratories, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. Received for publication January 27, 1998; accepted August 24, 1998 (8A4584). © The Histochemical Society, Inc.

KEY WORDS metalloporphyrins time-resolved luminescence microscopy phosphorescence autofluorescence FISH bleaching tyramide signal amplification

tion sensitivity of fluorescence-based microscopical methods. Furthermore, cytochemical amplification systems, such as the tyramide signal amplification (TSA) system, can provide an additional improvement of sensitivity (Bobrow et al. 1989; Adams 1992; Raap et al. 1995). Several studies have proved that TSA, a peroxidase-driven amplification system by which hapten-labeled radicals are created, can amplify the immunological signal at least 100-fold, depending on the detection method used (Adams 1992; Merz et al. 1995; de Haas et al. 1996; Werner et al. 1996). Despite these significant improvements, immunohistochemical detection of DNA, mRNA or antigens still can be strongly hampered by the presence of autofluorescence. Strictly speaking autofluorescence relates to the native fluorescence of cellular molecules. However, fixative-induced fluorescence and fluorescence of optical components such as the microscope objective and immersion oil also reduce the specific image contrast.

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de Haas, van Gijlswijk, van der Tol, Veuskens, van Gijssel, Tijdens, Bonnet, Verwoerd, Tanke

Using delayed luminescent labels bound to antibodies or nucleic acid probes, differentiation between the specific delayed luminescent signal and the fast decaying autofluorescence can be made, resulting in an enhanced image contrast (Marriott et al. 1991,1994; Seveus et al. 1992; Verwoerd et al. 1994). An example is the successful use of lanthanide chelates (Marriott et al. 1991,1994; Seveus et al. 1992), especially in combination with the TSA (de Haas et al. 1996). In a recent study we developed and evaluated metalloporphyrin immunoconjugates for application in timeresolved microscopy. Pt-coproporphyrin was found to be a suitable phosphorescent dye because of its high phosphorescence quantum yield, high molar extinction coefficient, moderate decay time, good photostability, and the possession of a large Stokes shift. These characteristics remained unchanged on conjugation to streptavidin and antibodies, and the biological activity of the labeled immunoconjugates was retained. These conjugates were successfully applied for detection of 28S rRNA in HeLa cells and CD4 epitopes on lymphocytes using a time-resolved laser scanning microscope (de Haas et al. 1997). Because such specialized equipment is rarely available, we therefore present in this article a new approach based on a time-resolved module, which is easily incorporated into a conventional microscope. Furthermore, we describe the preparation of immunoconjugates (streptavidin/antibodies) of Pd-coproporphyrin. Substitution of Pt by Pd results in a metalloporphyrin of relative longer lifetime. The synthesized Pt- and Pd-coproporphyrin conjugates were photophysically analyzed and evaluated for time-resolved detection of various antigens present in strongly autofluorescent tissues. They were also used for the detection of DNA and mRNA using FISH. The new time-resolved module was successfully applied to directly visualize metalloporphyrins labeled immunoreagent and nucleic acids.

Materials and Methods Preparation of Palladium-Coproporphyrindi-N-Hydroxysuccinimide Ester Palladium-3,8,13,18-tetramethyl-21H,23H-porphine -2,7,12, 18-tetrapropionic-di-N-hydroxy-succinimide ester (PdCP-2NHS) was prepared using a previously reported procedure (de Haas et al. 1997). Briefly, 22 mg Pd-coproporhyrin was dissolved in 900 l DMF (dimethylformamide; Aldrich, Bornem, Belgium) and 130 l 1-hydroxybenzotriazol (Aldrich) (60 mg/ml, 444 mM) and 120 l 1,3-dicyclohexylcarbodiimide (Pierce; Rockford, IL) (35 mg/ml, 170 mM) were added. The resulting solution was agitated for 15 min at room temperature (RT), after which 66.8 l N-hydroxysuccinimide (50 mg/ml, 434 mM) was added. This reaction mixture was agitated for an additional 20 hr in the dark at RT. After centrifugation, the supernatant (PdCP-2-NHS es-

ter concentration 23 mM) was removed and divided into small aliquots and stored at 20C in the dark. The PtCP-2-NHS ester (20 mM) was prepared in a similar way.

Labeling of Streptavidin and Goat Anti-rabbit IgG Antibody with PdCP-2-NHS Ester Streptavidin (Rockland; Gilbertsville, PA) was dissolved in 1 NHS buffer (5 NHS buffer 0.250 M Na-phosphate, pH 8.0, 500 mM NaCl, and 25 mM EDTA, pH 8.0). The immunoaffinity-purified goat anti-rabbit IgG fraction (Rockland) was first diluted in PBS (10 mM phosphate buffer, pH 7.4, 150 mM NaCl), which was subsequently replaced by a 1 NHS buffer by gel filtration. The streptavidin and goat anti-rabbit IgG were labeled with various amounts of PtCP-2-NHS and PdCP-NHS ester (see Table 2). After labeling, the conjugates were purified by gel filtration using a PD-10 column (Pharmacia; Uppsala, Sweden) equilibrated with PBSE (10 mM phosphate buffer, pH 7.4, 150 mM NaCl, 5 mM EDTA) and stored at 20C. The porphyrin/protein ratio (F/P), yield and biological activity were measured as previously reported (de Haas et al. 1997). The conjugates were photophysically analyzed as described below.

Preparation and Purification of PdCP/PtCPCoproporphyrin–dUTP 5-(3-aminoallylaminedeoxyuridine triphosphate) (AAdUTP) was synthesized according to the method described by Langer et al. (1981). The AAdUTP was dissolved in doubledistilled water to a concentration of 100 mM and stored at 20C until further use. Typically, to 40 l (20 mM) AAdUTP, 40 l 5 NHS buffer and 82 l water was added. After mixing, 50 l (20 mM) PdCP-2-NHS or PtCP2-NHS ester was added (2.5-fold excess). The reaction mixture was incubated for 3 hr at 37C. The same reaction mixture but without the porphyrin-2-NHS ester served as negative control. The course of the reaction was followed by HPTLC (high-performance thin-layer chromatography) using 70:30 (v:v) ethanol:water as the eluent. After collecting the (Pd,Pt)CP-7-dUTP fractions, the eluent was evaporated by vacuum rotary evaporation until dryness. The porphyrin– dUTP was dissolved in 100 l TEA and purified for a second time by reverse-phase HPLC to remove as much free porphyrin as possible in the dUTP sample. The final purity of the PtCP and PdCP dUTP was 95% as estimated on the basis of the HPLC profiles.

Photophysical Characterization of Pd/Pt-Coproporphyrin-7–dUTP Pt-coproporphyrin-7-dUTP and Pd-coproporphyrin-7-dUTP were dissolved in a 50 mM phosphate buffer, pH 8.0, to an absorption of approximately 0.1. The absorption spectra were recorded on a D4-64 Beckman absorption spectrophotometer. The excitation and emission spectra of both porphyrin–dUTP derivatives were recorded on a SPEX Fluorlog 2 spectrophotometer (SPEX Industries; Edison, NJ), using


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Time-resolved Microscopy Using Pt/Pd Porphyrins 400 nm and 380 nm to excite PtCP-7-dUTP and PdCP-7dUTP, respectively. For the recording of excitation spectrum, the 670-nm emission for PtCP and 650-nm emission for PdCP were selected. Luminescence lifetimes were determined with an OMAIII (optical multichannel analyzer) (EG&G; Vaudreuil, PQ, Canada) setup using rhodamine B (Q1.0) (Aldrich) dissolved in the same buffer as the porphyrin samples. The measured spectra and calculated quantum yield were corrected for the detector sensitivity and lamp intensity. The photophysical characterization of PtCP-labeled and PdCP-labeled goat anti-rabbit IgG antibody was performed in a similar way.

Enzymatic Labeling of DNA Probes by Nick Translation One g of DNA was incubated for 2 hr at 15C in a final volume of 50 l containing 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 0.5 mg/ml bovine serum albumin (Organon Teknika; Turnhout, Belgium), 10 mM DTT, 5 ng DNAse I (Boehringer; Mannheim, Germany), 10 U DNA polymerase I (Promega; Leiden, The Netherlands), 40 M dATP, 40 M dCTP, 40 M dGTP, 8 M dTTP (all nucleotides were from Promega), and 40 M biotin-16-dUTP, 40 M fluorescein12-dUTP (with the addition of free PtCP to study its effect on the DNA polymerase activity) or porphyrin-dUTP at various concentrations (see Figure 2). After nick translation, the fluorescein- or biotin-containing probe was ethanol-precipitated and dissolved in hybridization mixture. The porphyrin-containing DNA probes were purified by gel filtration before ethanol precipitation and dissolved in hybridization mixture (see below).

Chemical Labeling of DNA with Pt/Pd-coproporphyrin-NHS Ester The centromere probe specific for chromosome 1 (pUC 1.77; Cooke and Hindley 1979) was labeled by nick translation as described above, except that dTTP was replaced by allylamine–dUTP (2 l, 1 mM AAdUTP), and BSA was not present in the nick-translation buffer. After 2-hr incubation at 15C, the reaction was stopped by adding 5 l 0.5 M EDTA (pH 8.0) to the reaction mixture. The probe was purified by gel filtration, ethanol-precipitated, and subsequently dissolved in 1 NHS buffer at 40C. To the probe solution 15 l PdCP-2-NHS (23 mM) or PtCP-2-NHS (20 mM) was added and incubated overnight at 40C. The probe was purified by gel filtration and ethanol-precipitated. The pellet was dissolved in 50 l hybridization mix ([60% formamide, 2 SSC (20 SSC is 0.3 M sodium citrate buffer, pH 7.4, 3 M NaCl), 50 mM sodium phosphate, pH 7.4, and 10% dextran sulfate].

DNA In Situ Hybridization For DNA in situ hybridization, the following probes were used: centromeric probes for chromosome 1 (pUC 1.77, target size 3000 kb) (Cooke and Hindley 1979), for chromosome 17 (p17H8, target size 500 kb) (Waye and Willard 1986); and for chromosome 9 (pHUR98, target size 100 kb (Waye and Willard 1989), a cosmid probe for chromo-

some X (target size 40 kb), and a cocktail of plasmid probes comprising a 15.8-kb part of the thyroglobulin gene (Landegent et al. 1985). These probes were labeled by standard nick translation (see above) using biotin-16–dUTP (Boehringer), PtCP-7–dUTP, or PdCP-7–dUTP. All probes were purified by gel filtration. The preparation of metaphase spreads and fluorescence in situ hybridization using biotinylated probes were performed according to Wiegant et al. (1991,1993) and de Haas et al. (1996). Various immunocytochemical methods were applied after washing with TBS to detect the formed biotinylated hybrid: (a) streptavidin–PdCP (F/P 4.3) or streptavidin–PtCP (F/P 5.6); (b) streptavidin– PdCP (F/P 4.3) or streptavidin–PtCP(5.6), biotinylated goat anti-streptavidin (1:100), streptavidin–PdCP(F/P 4.3) or streptavidin–PtCP(F/P 5.6); (c) tyramide signal amplification, horseradish peroxidase (HRP)-labeled streptavidin (1:500) (NEN Life Science Products; Boston, MA), biotin–tyramide (5 g/ml), streptavidin–PtCP (F/P 5.6), or streptavidin–PdCP (F/P 4.3). The conjugates were incubated for 30 min at 37C in a moist chamber and washed three times with TNT (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05% Tween-20) at RT. After the final TNT wash, the slides were counterstained with a DAPI (4,6-diamino-2-phenylindole dihydrochloride) solution (5 ng/ml) for 10 min at RT. The slides were then washed twice with TBS, dehydrated in ethanol, air-dried, and embedded before evaluation.

Quantitative Measurement of Hybridization Signals Phosphorescence measurements were made with a modified fluorescence microscope (Aristoplan; Leica, Oberkochen, Germany) equipped with a cooled CCD camera (Photometrics, Tucson, AZ; type CE-200) connected to a SUN 4-330GX workstation. The CCD camera used has a KAF-1400 CCD chip (1320 1035 pixels). For determination of the integrated phosphorescence of the hybridization spots, a useractive program was developed using SCIL image (TPD; TU Delft, the Netherlands). Interaction by the user consisted of selecting each cell by mouse operation. The integrated phosphorescence of the spots was determined and corrected for background measured in a ring around the spots at a distance of about twice the spot diameter. The integrated phosphorescence intensity was divided by the exposure time to obtain the photon flux per spot. Before measurements, all images were corrected for dark current and for uneven illumination using uranyl glass. Per slide, 30 randomly selected interphase nuclei were measured. The mean value of the highest phosphorescence intensity was arbitrarily set at 1000. All other values were calculated from that value.

Preparation of PtCP-labeled Oligonucleotide Complementary to the 28S rRNA Sequence One hundred g of a 40 mer-(oligonucleotide sequence 5-CGTTTG-ATT-TGC-TAC-TAC-CAC-CAA-GAT-CTG-CAC-CTGC-3) containing an amino linker (5) was dissolved in 1 NHS buffer, after which 20 l PtCP-2-NHS ester (20 mM) was added to the solution. The reaction mixture was incubated at 50C for 3 hr. The excess of PtCP-2-NHS ester was removed by gel filtration using 100 mM TEA as eluent. The PtCP-labeled oligonucleotide was purified by reverse-phase HPLC (Smart system; Pharmacia). The oligonucleotide-labeled


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PtCP fractions were collected and dried using rotatory evaporation and were subsequently dissolved in TE (10 mM TrisHCl, pH 8.0, 2 mM EDTA) and stored at 20C.

lands). Tissue sections stained by immunoreagents conjugated to other fluorophores were embedded in 25 l Vectashield (Vector; Burlingame, CA).

28S rRNA Detection Using an Oligonucleotide Labeled with PtCP

Immunocytochemical Staining of Glutathione Transferase

HeLa cells were grown on uncoated microscope object slides for 2 days in Petri dishes containing Dulbecco’s minimal essential medium supplemented with 10% fetal calf serum (without phenol red) at 37C in a 5% CO2 atmosphere. After culturing, the cells were briefly rinsed in PBS and fixed for 10 min in 5% formaldehyde with 4% acetic acid in PBS. The slides were then washed with PBS (twice for 5 min) and three times with 70% ethanol. They were stored in 70% ethanol at 4C until further use. Further treatment of the cells and in situ hybridization were preformed according to van der Corput et al. (in press). The 28S oligonucleotide and its sense form were labeled with Texas Red and served as positive and negative control, respectively.

Preneoplastic foci in rat the liver were formed by induction with 2-acetylaminofluorene. The rat liver was removed after perfusion under anesthesia, fixed with formaldehyde, and embedded in Technovit 8100, and 3-m tissue sections were cut. The tissue sections were dried for 2 hr at 37C. To reexpose the masked antigenicity the plastic sections were treated with 0.1% trypsin (Gibco; Grand Island, NY) in 0.1% calcium chloride (Aldrich), pH 7.8, at 37C for 10 min. The endogenous peroxidase activity was inactivated and endogenous biotin was blocked (for detailed procedure see below). After rinsing in TBS, the sections were incubated with rabbit anti-glutathione transferase diluted 1:50 in TNB [0.5 % (w/v)] blocking reagent (Boehringer) in TBS at 37C in a moist chamber and washed in TBS. For the control sections this incubation step was omitted. The following detection methods were performed: (a) fluorescein-labeled goat antirabbit antibody 3 g/ml (Vector); (b) TSA using biotin–tyramide; biotin deposits were detected with streptavidin–PtCP (F/P 5.6) (5 g/ml) or streptavidin–PdCP (F/P 1.1 and 4.3), or streptavidin–FITC (Vector). The sections were washed, dehydrated and embedded. Contrast values were determined as follows. For each experimental condition (type of preparation and type of staining), three serial sections were used for contrast measurements. For each section, two images of preneoplastic noduli were recorded and quantitatively analyzed. Within such GST 7-7-positive nodules, four areas were selected (comprising five to seven cells). From this selected window, the total fluorescence intensity was measured. This value was divided by the fluorescence intensity for a similarly sized window from a cytochemically negative area in the proximity of the positively stained nodules to obtain a contrast value. For each immunocytochemical variant, 25–35 contrast values were obtained, averaged, and the standard deviation was calculated.

Photostability of Streptavidin–PdCP, Streptavidin– PtCP, DNA–PtCP, and DNA–PdCP Under In Situ Conditions The photostability of PtCP and PdCP bound to streptavidin and DNA was investigated using slides stained for FISH. For streptavidin–PtCP (F/P 5.6) or streptavidin–PdCP (F/P 4.3), interphase nuclei were hybridized with a biotinylated centromere-specific probe for chromosome 1 and subsequently stained with these streptavidin conjugates. For DNA–PtCP/ PdCP, the same probe was nick-translated using PtCP or PdCP-7–dUTP. The slides were stored in the dark and embedded before the measurements. To avoid bleaching of the porphyrins, images of autofluorescence generated by blue excitation were used for focusing. The excitation time of the specimens was varied from 0 to 1000 sec, and images were recorded and stored on hard disk for further analysis. The bleaching of each centromeric spot was followed in time, whereby the intensity of the spot at t 0 was set at 100% after subtracting the background signal. Within one image, at least three stained nuclei were measured and the experiment was repeated twice. The data points were averaged and a bleaching curve was drawn by hand. On the basis of the obtained data points, the bleaching decay time was calculated assuming a monoexponential decay.

Tyramide Signal Amplification For the evaluation of DNA and mRNA in situ hybridization and for the demonstration of antigens in tissues, the biotinbased TSA was employed (de Haas et al. 1996). After extensive washing with TNT (four times for 10 min each), the deposited biotin was detected by incubation with a conventional fluorochrome or a metalloporphyrin-labeled streptavidin or antibody.

Embedding All tissue sections stained with PtCP or PdCP conjugates were embedded in 25 l glycerol:0.5 M phosphate buffer, pH 8.0 (9:1 v/v) containing 20 mM glucose and 10 U/ml glucose oxidase (Boehringer Mannheim; Almere, The Nether-

Immunohistochemical Detection of Prostate-specific Antigen (PSA) Five-m sections of paraffin-embedded human prostate were attached to APTS-treated glass slides. The sections were deparaffinized twice for 10 min in xylene, washed twice with 100% ethanol for 10 min to remove the xylene, and rehydrated through an ethanol series (90, 70, 50%). The endogenous peroxidase activity was blocked by incubating the section with 3% (v/v) hydrogen peroxide in methanol for 30 min. The sections were washed with TNT for 5 min and blocked for 30 min at 37C with TNB in a moist chamber and subsequently incubated with rabbit anti PSA (Dakopatts; Glostrup, Denmark) diluted 1:400 for 18 hr at 4C. They were washed in TNT, three times for 5 min, and various immunocytochemical detection methods were applied to the slides: (a) goat anti-rabbit–FITC (4 g/ml); (b) goat antirabbit–PtCP (5g/ml) (conjugates with different F/P ratios); (c) goat anti-rabbit–PdCP (5g/ml) (conjugates with differ-


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Time-resolved Microscopy Using Pt/Pd Porphyrins ent F/P ratios); (d) HRP-labeled goat anti-rabbit after which the TSA system was applied (see above). The deposited biotin was detected with streptavidin–PTCP (F/P 5.6) or streptavidin–PdCP (F/P 4.3) or streptavidin–FITC (Dakopatts). The sections were dehydrated and embedded.

Detection of Wild-type p53 in Rat Liver Tissue The detection of wild-type p53 was performed according to van Gijssel et al. (1997,1998). The TSA system was applied to the tissue using biotin–tyramide (see above). The deposited biotin was subsequently detected by streptavidin–PtCP (F/P 5.6), streptavidin–PdCP (F/P 4.3), or streptavidin–FITC (Vector), after which the slides were embedded.

Immunohistochemical Detection of the Androgen Receptor in Prostate Tissue Preparation and endogenous peroxidase inactivation were as described for PSA detection. The paraffin-embedded prostate tissue sections were immunostained for the androgen receptor according to Ruizeveld de Winter et al. (1990). After incubation with HRP-labeled rabbit anti-mouse, the TSA was performed (see above) using biotin–tyramide as substrate. The deposited biotin was detected using streptavidin– PtCP (F/P 5.6) or streptavidin–PdCP (F/P 4.3). The sections were washed, dehydrated, and embedded (see above).

Immunohistochemical Detection of Glucagon in Human Pancreas Tissue Five-m paraffin-embedded sections of human pancreas tissue (kindly provided by P. Janssen; Department of Pathology, Erasmus University, Rotterdam, The Netherlands) were deparaffinized, rehydrated, and peroxidase-inactivated (see above). They were blocked with TNB for 30 min at 37C and incubated with the rabbit anti-glucagon antibody according to the instructions of the manufacturer (Dakopatts), washed with TNT for three times for 5 min, and incubated with goat anti-rabbit–PtCP or goat anti-rabbit–PdCP conjugates with various F/P ratios. In addition, the sections were incubated with HRP-labeled goat anti-rabbit, after which TSA was performed (see above). The deposited biotin was detected using streptavidin–PdCP (F/P 4.3), streptavidin–PtCP (F/P 5.6), or streptavidin–FITC, after which the tissue sections were dehydrated and embedded.

Detection of Chalcone Synthase (chs A) mRNA Transcripts in the Corolla of Petunia hybrida by RNA In Situ Hybridization Petunia hybrida variety V26 was used in this study. The gene chalcone synthase A (chsA) is highly expressed in the corolla of young flowers (Koes et al. 1989). Corollas were fixed in 4% formaldehyde and 5% acetic acid by vacuum infiltration for 5 min, followed by an incubation in fresh fixative for 30 min on ice. The tissue was dehydrated through an ethanol series and embedded in Paraplast. The embedded corolla tissue was cut into 12-m sections, stretched on a drop of DEPC-treated water on poly-l-lysine-coated slides, and dried overnight at 37C. The chsA coding region cloned into VIP143 and VIP131 was used to generate labeled sense and anti-sense riboprobes, respectively (van Blokland et al. 1994).

The plasmids were linearized and served as template for the synthesis of digoxigenin-11–dUTP by in vitro transcription according to the manufacturer’s instructions (Boehringer Mannheim). The riboprobes were subjected to alkali hydrolysis to generate fragments of an average size of 150 nucleotides to allow better tissue penetration. For each probe, the amount of incorporated label was determined and the probes were stored in DEPC treated water at a concentration of 5 ng/l. In situ hybridization was as described by Canas et al. (1994). Briefly, the sections were deparaffinized in xylene and rehydrated by passing through an ethanol series and water. The sections were treated with 1 g/ml proteinase K in 50 mM Tris-HCl, pH 7.5, for 20 min at 37C and 0.25% acetic anhydride in 85 mM TEA buffer, pH 8.0, for 10 min at RT. Next, the sections were dehydrated through an ethanol series and air-dried. Hybridization was carried out at 45C overnight with 10 l of hybridization solution. This hybridization solution contained 0.5 ng/l digoxygenin-labeled RNA, 45% formamide, 300 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 Denhardt’s solution, 10% dextran sulfate, 10 mM DTT, 250 ng/ml tRNA, and 100 g/ml poly(A). After hybridization, sections were rinsed briefly in 2 SSC to allow the coverslips to fall off and then incubated three time for 25 min at 55C in 0.2 SSC. Subsequently, an RNase A treatment (20 g/ml in 500 mM NaCl, TE pH 8.0) was performed at 37C for 30 min. The sections were rinsed in TNT three times for 10 min at RT, blocked with TNB at 37C for 30 min, and various immunocytochemical detection methods were performed: (a) alkaline phosphatase-labeled sheep anti-digoxygenin (1:1000) (Boehringer Mannheim), and developed with NBT/BCIP as substrate for 15-20 hr; (b) sheep anti-digoxygenin–FITC (Boehringer Mannheim); (c) biotinylated mouse anti-digoxin (1:200) (Sigma; St Louis, MO) and streptavidin–PtCT, streptavidin– PdCP, or streptavidin–FITC respectively; (d) TSA (see above) using biotin–tyramide; the deposited biotin molecules were stained with streptavidin–FITC, streptavidin–PtCP (F/P 5.6), or streptavidin–PdCP (F/P 4.3). After staining the sections were washed, dehydrated, and embedded.

Conventional Microscopy Photomicrographs were taken with a Leica DM epifluorescence microscope equipped with a 100-W mercury arc lamp and appropriate filter sets for visualization of DAPI fluorescence and porphyrin phosphorescence. The filter block for both porphyrins contained a bandpass 500–560-nm excitation filter, a 580-nm dichroic beam splitter, and a 600–700nm bandpass filter for selection of the emission. A 40 NA 1.30, a 100 1.30 PL Fluortar, or a 63 NA 1.32 PL APO objective was used, using 640 ASA 3M color slide film. The exposure times were 2–5 sec (DAPI counterstain) and 2–5 min (porphyrin).

Time-resolved Microscopy Delayed imaging was accomplished using a special module incorporated into the DM microscope. It consists of a minichopper mounted in the illumination block, which contains the excitation shutter and diaphragm. A polarizer (Ferro Liquid Crystal type) was placed in a standard filter holder and mounted at the emission side (below the tune lens to al-


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low direct visualization of delayed luminescence when needed). An electronic control unit was constructed to provide synchronized excitation and detection of delayed luminescence for dyes with lifetimes higher than 50 s. The system suppresses fluorescence of shorter lifetimes by at least 1000-fold.

Results Photophysical Properties of PtCP and PdCP

The photophysical properties of free PtCP or of PtCPbound to streptavidin or antibodies have been determined previously (de Haas et al. 1997). Figure 1 shows the excitation and emission spectra and structure of PdCP, showing excitation maxima at 376 nm, 504, and 545 nm, and an emission maximum at 760 nm. At 376 nm, PdCP has a molar extinction coefficient of 72,000 M1cm1. Conjugation to proteins (antibodies or streptavidin) or allylamine–dUTP results in a shift of 14 nm towards the red part of the spectrum. Furthermore, the replacement of platinum by palladium into the porphyrin ring structure increases the lifetime by a factor of 10 to about 1000 s (see Table

1). The quantum yield of PdCP was threefold lower than that of PtCP. As expected, the lifetime of PdCP is strongly influenced by the presence of oxygen and by the viscosity of the medium. Vectashield, a frequently used anti-fading reagent for FISH and immunofluorescence, was found to decrease the quantum yield and the lifetime of PdCP, regardless of the presence or absence of oxygen. Several streptavidin and goat anti-rabbit IgG conjugates labeled with PdCP were analyzed. PdCP showed almost no nonspecific adsorption to streptavidin or immunoglobulins. After two rounds of purification by gel filtration, the resulting F/P ratio was less than 0.02 starting with a tenfold excess of porphyrin. Table 2 shows the resulting photophysical data of the prepared conjugates. A PdCP/protein ratio higher than 10 resulted in a decreased yield and significant loss of biological activity. For antibodies this critical F/P value was higher (F/P 8.3). For streptavidin, we observed that an increased F/P ratio leads to a decreased average quantum yield of the PdCP molecule, a phenomenon that was less prominent for antibodies. The optimal F/P ratio was 4.3; higher F/P ratios re-

Figure 1 (A) Excitation and emission spectra of PdCP (bold), streptavidinPdCP (coarse dotted line), and PdCP– dUTP (fine dotted line). (B) Molecular structure of PdCP. (C) Molecular structure of PtCP-7–dUTP.


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Time-resolved Microscopy Using Pt/Pd Porphyrins Table 1 Photophysical properties of PtCP, PdCP,PtCP-7–dUTP, PdCP-7–dUTPa in PB O2 and Q in PBO2 and Q in PBG O2 and Q in PBGO2 and Q in PBGLOX O2 and Q in Vectashield O2 and Q

PtCP

PdCP

PtCP-7–dUTP

2 (0.04) 52 (0.23) ND 92 (0.54) 91 (0.55) 9 (0.04)

A (0.01) A (0.01) 165 (0.03) 1100 (0.17) 1130 (0.17) 11 ( 0.01)

A (0.02) 86 (0.38) 56 (0.25) 94 (0.47) 97 (0.42) 11 (0.04)

PdCP-7–dUTP A ( 0.01) A (0.11) 178 (0.04) 1150 (0.18) 1190 (0.19) 12 ( 0.01)

a ,

decay time; O2, oxygen present; O2, oxygen absent; PB, 50 mM phosphate buffer; PBG, glycerol:0.5 M PB (9:1 v/v), pH 8.0; PBGLOX, glycerol:0.5 M PB (9:1), pH 8.0 containing 20 mM glucose and 20 U/ml glucose oxidase; Q, quantum yield; ND, not determined; A, absorption too low; the decay time is given in sec; the error in the decay time is 5%; the error in the quantum yield is 0.02.

sulted in a significant reduction of F/P Q value, which is an indication of the overall quantum yield of a particular conjugate. As an exception, no decrease in F/P Q value was found for the labeled goat anti-rabbit antibodies. At an F/P ratio of 8.3, the achievable yield and biological activity were significantly affected. Higher F/P ratios of antibody conjugates are therefore not considered to be useful. Preparation of PtCP-7–dUTP and PdCP-7–dUTP

A threefold excess of PtCP-NHS ester was found optimal at 2-hr reaction time to convert AA–dUTP into PtCP-7–dUTP and PdCP-7–dUTP (for structure see Figure 1) at 2-hr reaction time. Photophysical properties of PtCP and PdCP-labeled nucleotides are given in Table 1. The quantum yield and lifetime of PtCP and PdCP did not change very much on conjugation. An increase in viscosity by the addition of glycerol resulted in an increase of quantum yield and lifetime, probably as a result of decreased oxygen diffusion in the embedding solution. In addition, the molar extinction coefficient of both porphy-

rins did not change very much upon covalent conjugation to allylamine–dUTP. The removal of oxygen by the glucose/glucose oxidase system proved to be as efficient as flushing with argon gas. Glucose oxidase itself did not influence the quantum yield and lifetime. The photophysical characteristics of the porphyrin– dUTPs strongly changed when Vectashield was used as anti-fading agent. PtCP-7–dUTP and PdCP-7–dUTP were then tested as a substrate for DNA polymerase to prepare a phosphorescent-labeled DNA probe. First, a possible inactivation of DNA polymerase activity by the porphyrin moiety was investigated. Standard nick translations were performed using fluorescein-12–dUTP as label with the addition of various amounts of PtCP. The probe was hybridized and the signal intensity of the spots was measured (Figure 2). We found that PtCP affects the DNA polymerase activity only at PtCP concentrations higher than 150 M. In a standard nick translation, such a concentration will not be reached. The nick translation was optimized by applying various dTTP/PtCP–dUTP ratios into the nick-translation medium. The labeled probes were hybridized and

Table 2 Photophysical properties of streptavidin and goat anti-rabbit antibody labeled with PdCP-2–NHS ester under various conditionsa Conjugate Streptavidin Streptavidin Streptavidin Streptavidin Streptavidin Streptavidin GaR GaR GaR GaR GaR GaR GaR

PdCP-2–NHS/protein ration

Obtained F/P ratio

Yield in %

Activity in %

-O2 in sec

QQ2

F/PQ

2 4 8 10 15 20 2 4 6 8 10 15 20

0.9 1.7 4.3 5.2 6.4 7.9 1.1 1.7 3.0 3.8 5.2 6.2 8.3

95 95 95 90 70 60 95 95 95 95 95 95 80

90 90 90 90 90 80 90 90 90 90 90 90 70

ND 1000 810 884 835 759 1370 920 1020 1060 940 930 ND

0.11 0.09 0.11 0.05 0.03 0.07 0.08 0.08 0.08 0.06 0.06 0.06 ND

0.19 0.39 0.57 0.32 0.24 0.08 0.14 0.24 0.30 0.31 0.37 0.50 ND

a The proteins were labeled at a concentration of 2 mg/ml; PB, 50 mM phosphate buffer; the conjugates were measured in glycerol:PB (9:1 v/v); GaR, goat anti-rabbit antibody; Q, quantum yield; , decay time; O2, oxygen absent due to the addition of glucose/glucose oxidase (20 mM glucose and 20 U/ml glucose oxidase); ND, not determined; the error in the yield is 5%; the error in the quantum yield is 5%; the error in the F/P ratio is 0.2.


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Figure 2 Optimization of the nick translation for probe labeling with PtCP. (A) Optimization of PtCP/dTTP ratio during the nicktranslation labeling (See text for further details). (B) The influence of free PtCP on the DNA polymerase activity. pUC 1.77 was labeled by nick translation in the presence of various amounts of free PtCP. The labeled probes were purified and used for hybridization experiments, after which the probe intensity was measured.

the intensity of the spots was measured. Maximal intensity was achieved for 30–90% of PtCP–dUTP (Figure 2). The signal intensity of the directly labeled probe pUC 1.77 with (Pt/Pd)CP using chemical or enzymatic methods was compared. Chemical labeling of this probe (after incorporation of allylamine–dUTP) provided the brightest spots (approximately 1.7-fold compared to enzymatic labeling) (Figure 3). Similar results were obtained when this experiment was performed with fluorescein–NHS ester (data not shown). This increase in signal intensity is explained by a higher porphyrin or fluorescein density in the probe. Use of Streptavidin–(Pt/Pd)CP, (Pt/Pd)CP-7–dUTP for DNA In Situ Hybridization

Streptavidin–(Pt/Pd)CP and (Pt/Pd)CP-7–dUTP were evaluated for DNA in situ hybridization for the detection of DNA sequences of various sizes (Table 3). The

Figure 3 The relative signal intensities after DNA in situ hybridization using pUC 1.77. (1) Labeled with PdCP-7–dUTP using nick translation. (2) Labeled with PtCP-7–dUTP using nick translation. (3) Labeled with PtCP-2–NHS after enzymatic incorporation of allylamine–dUTP. (4) Indirect detection using a biotinated probe (nick translation) detected with streptavidin–PtCP (F/P 5.6).

sensitivity of the directly (enzymatically) labeled DNA probes was limited because only large targets could be visualized. The TSA detection method provided intense bright signals for each target, even in the case of thyroglobulin gene detection (target size 15 kb) (Figure 4). Each signal detected by digital fluorescence microscopy or by eye was also visible using the timeresolved microscope in the delayed mode. Detection of 28S rRNA in HeLa Cells using a PtCP-labeled Oligonucleotide

Figure 5 shows the HPLC chromatogram for the purification of the 28S PtCP-labeled oligonucleotide. Owing to the hydrophobicity of PtCP, a higher percentage of acetonitrile was found necessary to elute the labeled oligonucleotide from the column compared to the un-

Table 3 Evaluation of Pt/PdCP-7–dUTP and streptavidin–(Pt/Pd)CP for detection of DNA sequences on metaphase spreads using DNA in situ hybridizationa Detection method Probe directly labeled with PtCP/PdCP Probe labeled with biotin and detected with one layer sAV-PtCP/sAV-PdCP Probe labeled with biotin and detected with 2 layers sAV-PtcP/sAV-PdCP Tyramide signal amplification

p1 (3000 KB)

p17 (500 KB)

p9 (100 KB)

Cosmid (40 KB)

Thyroglobulin gene (15.8 KB)

( )/( ) ( )/( )

( )/( ) ( )/( )

()/() ( )/( )

()/() ()/()

()/() ()/()

( )/( )

( )/( )

( )/( )

( )/()

( )/()

( )/( )

( )/( )

( )/( )

( )/( )

( )/( )

/ sign indicates the presence or absence, respectively, of a specific hybridization signal. Signal intensities increased from directly labeled probe to probes demonstrated by TSA (tyramide signal amplification).

aA


191

Time-resolved Microscopy Using Pt/Pd Porphyrins

Figure 4 Detection of the 15-kb part of the thyroglobulin gene using TSA (biotin) and streptavidin–PtCP (F/P 5.6). Bar 5 m.

labeled oligo. A 50-fold excess of PtCP-2–NHS resulted in a labeling percentage of at least 90%. For detection of 28S rRNA in HeLa cells, the Texas Red-labeled oligonucleotide gave strong cytoplasm staining (see Figure 6). The PtCP-labeled probes gave a similar type of staining compared to the Texas Redlabeled probes, although the intensity of the PtCP was somewhat lower (Figure 6). No signal was detected when the Texas Red- or PtCP-labeled sense probe was used, which confirmed the specificity of the hybridization procedure. Bleaching

PdCP was found to show a higher bleaching rate than PtCP (Figure 7). This may be explained by the longer lifetime of the triplet state of PdCP, which increases the chance of T1–T1 and T1–S0 interactions, which are known to result in photochemical destruction of the phosphorescent molecule. Furthermore, we found that PtCP directly bound to the DNA probe bleached more rapidly than when bound to an antibody. Detection of Glucagon in the Islets of Langerhans

The staining of glucagon in A-cells of the islets of Langerhans using fluorescein-labeled goat anti-rabbit shows that the A-cells are dispersed throughout the is-

Figure 5 HPLC purification of 40-mer 28S oligonucleotide labeled with PtCP. The numbers shown indicate time (min) after injection. The 28S oligo-PtCP elutes at 27.1 min.

lets, although there is a tendency for most A-cells to be found at the periphery of an islet. Strong autofluorescence of the pancreas tissue and small strongly autofluorescent spots were observed. To improve the contrast of the staining, TSA (biotin) was employed using fluorescein-labeled streptavidin to detect the deposited biotin molecules. An increased staining for glucagon was observed (Figure 8), with a higher contrast compared to the conventional staining. Using time-resolved microscopy with PtCP- or PdCP-labeled streptavidin after TSA (biotin), further improvement of the image contrast was obtained owing to the suppression of autofluorescence (Figure 8). The intensity of the PdCP staining was lower compared to the PtCP staining. For the PtCP- and PdCP-labeled secondary antibodies (goat anti-rabbit) with various F/P ratios, specific glucagon staining was observed only when the microscope was set in the delayed mode. The staining intensity of glucagon increased with F/P ratio, reaching a plateau of 5.2 for PdCP and 6.1 for PtCP; higher F/P ratios did

Figure 6 Staining of 28S rRNA in HeLa cells. (A) Using a Texas Redlabeled oligonucleotide (red), DAPI counterstain in blue. (B) DAPI counterstaining. (C) Using 28S PtCP-labeled oligonucleotide; microscope in the delayed mode (integration time 30 sec). Bar 5 m.


192

de Haas, van Gijlswijk, van der Tol, Veuskens, van Gijssel, Tijdens, Bonnet, Verwoerd, Tanke F/P 5.6 had the highest contrast value, e.g., 17.2, which is fourfold more than streptavidin–FITC. For streptavidin–PdCP, such a high value was not obtained because PdCP possesses a tenfold long lifetime than PtCP, thereby producing tenfold fewer photons at saturation conditions. Liver sections of rats that were not treated (negative control) showed no staining. Detection of p53 in Liver Tissue

Figure 7 Bleaching of streptavidin–PtCP, streptavidin–PdCP, DNA probe labeled with PtCP and PdCP. The stained centromeric region of chromosome 1 was used for measurements. The relative error in the intensity is 10–25%; the 95% confidence interval for the bleaching decay time is given in the figure.

not increase the staining intensity. No staining was observed when the primary anti-glucagon antibody was omitted from each particular detection method.

Liver sections of N-OH–AAF-treated rats showed positive staining for p53 (Figure 11) utilizing TSA (biotin) after the ABC method detected with fluoresceinlabeled streptavidin. p53 was localized in the nucleus, as shown by DAPI counterstaining. Detection of TSAdeposited biotin by PtCP-labeled streptavidin resulted in darkly stained red nuclei against a strongly autofluorescent background. The use of the time-resolved module completely suppressed this background, showing more positive nuclei than were detectable with conventional microscopy. In addition, the PdCP-labeled streptavidin conjugate yielded positive staining, although the brightness was lower compared to the PtCP staining. No staining was found in liver sections of control animals or when the primary antibody was omitted.

Detection of Glutathione Transferase 7-7

Contrast measurements were performed on rat liver sections that were stained for GST 7-7 and embedded in plastic (Figure 9). The advantage of plastic embedding is the preservation of the ultrastructural morphology. The suppression of the autofluorescence using the time-resolved module in the delayed mode yielded the highest contrast values (Figure 9) (for images see Figure 10). In the continuous mode, PtCPand PdCP-labeled streptavidin provided a lower image contrast than was achievable with fluorescein-labeled streptavidin ( 2), since they produce less photons per time unit because of their long lifetime. In the delayed mode, however, the streptavidin–PtCP conjugate with

Detection of Prostate-specific Antigen

The strong autofluorescence of the stroma results in a poor contrast for PSA staining using a fluoresceinlabeled secondary antibody. In addition, many strong autofluorescent spots impeded objective evaluation of the staining. The contrast significantly increased when TSA (biotin) was employed using fluorescein-labeled streptavidin to detect the deposited biotin (Figure 12). The staining with PtCP-labeled streptavidin (after TSA) resulted in a darkly red stained lumen, whereas the autofluorescence of the stroma was more orangelike. In this situation, the highest contrast was obtained using the time-resolved module (Figure 12).

Figure 8 Detection of glucagon in paraffin-embedded sections of human pancreas tissue. (A) Using TSA (biotin) and streptavidin–FITC. (B) Using TSA (biotin) and streptavidin–PtCP (F/P 5.6). (C) Digitized image similar to B using the time-resolved microscope in the conventional mode. (D) Digitized image similar to C using the microscope in the delayed mode (integration time 30 sec). Bar 10 m.


193


Figure 9 Contrast values for the immunohistochemical detection of GST 7-7 in plastic-embedded rat liver using various detection methods. (1) GaR–FITC; (2) GaR–HRP/TSA(biotin)/Strav–FITC; (3) GaR-HRP/TSA(biotin)/Strav–PtCP(F/P 5.6); (4) GaR-HRP/TSA(biotin)/ Strav–PdCP(F/P 0.9); (5) GaR–HRP/TSA(biotin)/Strav–PdCP(F/P 4.3). GaR–FITC, fluorescein-labeled goat anti-rabbit; GaR–HRP, peroxidaselabeled goat anti-rabbit; Strav–FITC, fluorescein-labeled streptavidin; TSA (biotin), tyramide signal amplification using biotin–tyramide as substrate.

PdCP-labeled streptavidin instead of PtCP resulted in a similar staining, although the intensity was lower. Specific staining of PSA was also obtained using the PtCP- and PdCP-labeled secondary goat anti-rabbit antibody conjugates. The obtained intensity and contrast were less compared to the TSA-based staining procedure. Omitting the primary rabbit anti-PSA antibody revealed no staining. Detection of Human Androgen Receptor in Prostate Tissue

The strong autofluorescence of the stroma and secretions makes it difficult to visualize the nuclear androgen receptor using PtCP-labeled streptavidin (after performing TSA) (Figure 13) with conventional microscopy. The contrast was improved by time-resolved microscopy, and the nuclei of basal cells, secretory cells, fibroblasts, and smooth muscle cells of the prostate tissue were found to be positively stained for the androgen receptor. The staining intensity of streptavidin–PdCP after TSA (biotin) was weak, and long integration times were required for visualization of the receptor. By omitting the Fab 39.4 antibody, no nuclear staining was observed. Detection of Chalcone Synthase mRNA in Petunia hybrida

To confirm the hybridization procedure, probe specificity, and expression pattern of chalcone synthase A (chsA) mRNA before fluorescence microscopic detec-

tion, digoxygenin-labeled anti-sense and sense probes were hybridized and detected using alkaline phosphatase-labeled sheep anti-digoxygenin. NBT/BCIP was used as substrate for alkaline phosphatase and produces a precipitate that was evaluated under brightfield microscopy (Figure 14).The highest staining with the anti-sense RNA probe was observed at the upper epidermis of the corolla. In addition, the lower epidermis showed some chsA mRNA expression. The intermediate tissue layer showed no chsA mRNA expression. No staining was observed with the sense RNA probe. Conventional immunofluorescence detection of chsA mRNA transcripts using fluorescein-labeled antidigoxygenin failed because of the high autofluorescence present in the corolla. The application of TSA (biotin) demonstrated the presence of transcripts, although the use of fluorescein-labeled streptavidin still resulted in a poor contrast (Figure 14). PtCP-labeled streptavidin yielded a strongly improved contrast after switching the time-resolved module to the delayed mode. An identical chsA mRNA expression pattern was found as for the alkaline phosphatase NBT/BCIP detection. Using PdCP-labeled streptavidin, a similar expression pattern was observed with an improved contrast compared to fluorescein-labeled streptavidin. The use of a sense probe under the same conditions revealed no staining.

Discussion We recently reported on Pt-coproporphyrin-labeled antibodies and streptavidin for time-resolved microscopy (de Haas et al. 1997). Pt-coproporphyrins are suitable delayed luminescent labels because of their high phosphorescence quantum yield, moderate triplet lifetime (70–90 sec), high molar extinction coefficient, and good photostability. On conjugation to antibodies or streptavidin these properties are retained. In addition, Pt-coproporphyrin can be used in combination with other fluorochromes, since they do not require the use of a special embedding medium as do the lanthanide chelates (Merckoglass) in which conventional fluorophores hardly fluoresce (de Haas et al. 1996). In this study we tested platinum and also palladium coproporphyrin-labeled antibodies and streptavidin for the detection of various antigens using a time-resolved module which is easily incorporated into a conventional fluorescence microscope. In our previous work we used a time-resolved laser microscope with an argon ion laser as light source that emits at 514 nm, despite the fact that PtCP has a low molar extinction coefficient at this wavelength. In the present study, a mercury arc lamp is used as excitation source, which strongly emits at 546 nm, close to the excitation maximum of PtCP. Tissue sections were embedded in glyc-


194


Figure 10 Detection of GST 7-7 in plastic-embedded tissues. (A) Digitized image using the time-resolved microscope in the conventional mode using TSA (biotin) and streptavidin–PtCP (F/P 5.6) for detection. (B) Digitized image identical to A using the microscope in the delayed mode (integration time 60 sec). Bar 10 m. Figure 11 Detection of p53 in paraffin-embedded rat liver tissue. (A) Using TSA (biotin) after the ABC method and streptavidin–FITC. (B) Using TSA (biotin) after the ABC method and streptavidin–PtCP (F/P 5.6); digitized image using the microscope in the conventional mode. (C) Digitized image identical to B using the microscope in the delayed mode (integration time 30 sec). Bar 10 m. Figure 12 Detection of prostate-specific antigen in paraffin-embedded sections of human prostate tissue. (A) Using TSA (biotin) and streptavidin–FITC. (B) Using TSA (biotin) and streptavidin–PtCP (F/P 5.6). (C) Digitized image using the time-resolved microscope in the conventional mode. Digitized image similar to B using the microscope in the delayed mode (integration time 15 sec). Bar 10 m.


195


erol/phosphate buffer containing glucose/glucose oxidase, for two purposes. First, because triplet quantum states are sensitive for oxygen, removal of oxygen avoids a shorter lifetime and reduces phosphorescence quantum yields. Next, a secondary effect of this embedding medium is that the generation of oxygen radicals, which are believed to cause bleaching, is prevented. Commonly used anti-fading reagents such as DABCO, Slowfade, and para-phenylene diamine (Florijn et al. 1995) cannot be used because these anti-fading reagents apparently contain triplet quenchers which, as we noted, affected the lifetime and quantum yield of Pt- and Pd-coproporphyrin. Various antigens were detected by immunohistochemistry using streptavidin and goat anti-rabbit labeled with Pt/Pd-coproporphyrin using time-resolved microscopy. These antigens have in common that they occur in generally strong autofluorescent tissue e.g., PSA and the androgen receptor in prostate tissue, glucagon in pancreas tissue, p53 and GST 7-7 in liver. The model for RNA FISH, e.g., chalcone synthase A mRNA, is present in the corolla of Petunia hybrida. Detection of these antigens and this particular mRNA was also performed using the tyramide system and a comparison was made. In general, the highest contrast for these antigens and mRNA was obtained using the TSA system with biotin as substrate. The obtained contrasts were better than when fluorescein-labeled immunoreagents were used. Specific staining was also obtained with PdCP- and PtCP-labeled goat anti-rabbit, although the acquired signal intensity was lower than for the TSA system. This is explained by the large amplification that can be obtained using the TSA system (Merz et al. 1995; Raap et al. 1995; Werner et al. 1996). For PdCP, we found conjugates with F/P 4.3 for streptavidin to be optimal and for antibodies with F/P 6.3. These values are in good agreement with the photophysical data of the conjugates. For phosphorescent labeling of DNA probes, we synthesized PtCP and PdCP dUTPs and found that they retained their quantum yield, lifetime, and molar extinction coefficient compared to nonmodified PtCP and PdCP. Both phosphorescent dUTPs were incorporated with the same efficiency as other fluorescent

dUTPS, such as fluorescein-12–dUTP, using DNA polymerase. PtCP was also accepted by terminal deoxynucleotidyl transferase to end-label oligonucleotides (data not shown). However, enzymes that are used for the PCR reaction to produce large amounts of labeled DNA probes did not accept these Pt/Pd–dUTPs. The polymerases were most likely inactivated by the porphyrin molecule, because it is known that heme (a porphyrin derivative) as occurs in blood samples negatively affects the PCR reaction. Alternatively, for the labeling of PCR fragments, first allylamine–dUTP (AA-dUTP) should be incorporated, which then can be labeled with the Pt/PdCP–NHS ester.Various DNA probes were directly labeled with these dUTPs by nick translation and the sensitivity of these probes in FISH is currently sufficient to detect centromeric repeat sequences. Using indirect detection, single-copy gene detection is feasible, as shown for the thyroglobulin gene. The detection of 28S rRNA using oligonucleotides labeled at the 5 end and with coproporphyrins shows that this type of FISH technique is possible. The detection of DNA sequences in autofluorescent tissue sections is currently under study. Finally, the timeresolved module described and used in this study is a low-cost option to directly visualize the phosphorescence of the coproporphyrin. In addition, the modular character also allows selection of the conventional fluorescence mode using the same microscope hardware. Acknowledgments Supported by Technology Foundation (NWO, The Netherlands) grant LGN 22.2734 and in part by Boehringer Mannheim (Penzberg, Germany) and Leica (Wetzlar, Germany).

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Figure 13 Detection of the human androgen receptor in paraffin-embedded prostate tissue. The detection was performed using TSA (biotin) and streptavidin–PtCP (F/P 5.6). (A) Digitized image using the time-resolved microscope in the conventional mode. (B) Using the microscope in the delayed mode (integration time 120 sec). Bar 10 m. Figure 14 Detection of chalcone synthase A mRNA transcripts in the corolla of Petunia hybrida by RNA in situ hybridization. (A) Digoxygenin-labeled RNA anti-sense probe detected with sheep anti-digoxygenin–AP developed with NBT/BCIP. Note that chsA is expressed in the upper epidermis of corolla tissue (brown precipitate). The tissue section is counterstained with toluidine blue. (B) Similar to A using the sense probe (negative control). (C) Digoxygenin-labeled RNA anti-sense probe detected with TSA (biotin) and streptavidin–FITC. (D) Digoxygenin-labeled RNA anti-sense probe detected with TSA (biotin) and streptavidin–PtCP(F/P 5.6) using the time-resolved microscope in the conventional mode. (E) Identical to D using the microscope in the delayed mode (integration time 40 sec). Bar 10 m.


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