ARTICLE |
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.
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Summary |
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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 modular- based time-resolved microscope was constructed and used for the evaluation of porphyrin-stained 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 47:183196, 1999)
Key Words: metalloporphyrins, time-resolved luminescence microscopy, phosphorescence, autofluorescence, FISH, bleaching, tyramide signal amplification
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Introduction |
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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 (
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.
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 (
In a recent study we developed and evaluated metalloporphyrin immunoconjugates for application in time-resolved 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 (
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.
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Materials and Methods |
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Preparation of Palladium-Coproporphyrin- di-N-Hydroxysuccinimide Ester
Palladium-3,8,13,18-tetramethyl-21H,23H-porphine-2,7,12, 18-tetrapropionic-di-N-hydroxy-succinimide ester (PdCP-2- NHS) was prepared using a previously reported procedure (
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 x NHS buffer (5 x 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 x 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).
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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 (
Preparation and Purification of PdCP/PtCP-CoproporphyrindUTP
5-(3-aminoallylaminedeoxyuridine triphosphate) (AAdUTP) was synthesized according to the method described by
Photophysical Characterization of Pd/Pt-Coproporphyrin-7dUTP
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 porphyrindUTP derivatives were recorded on a SPEX Fluorlog 2 spectrophotometer (SPEX Industries; Edison, NJ), using 400 nm and 380 nm to excite PtCP-7-dUTP and PdCP-7-dUTP, 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 (Q=1.0) (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 µg of DNA was incubated for 2 hr at 15C in a final volume of 50 µl 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 µM dATP, 40 µM dCTP, 40 µM dGTP, 8 µM dTTP (all nucleotides were from Promega), and 40 µM biotin-16-dUTP, 40 µM fluorescein-12-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).
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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;
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) (
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 x 1035 pixels). For determination of the integrated phosphorescence of the hybridization spots, a user-active 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 µg of a 40 mer-(oligonucleotide sequence 5'-CGT-TTG-ATT-TGC-TAC-TAC-CAC-CAA-GAT-CTG-CAC-CTG-C-3') containing an amino linker (5') was dissolved in 1 x NHS buffer, after which 20 µl 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 PtCP fractions were collected and dried using rotatory evaporation and were subsequently dissolved in TE (10 mM Tris-HCl, pH 8.0, 2 mM EDTA) and stored at -20C.
28S rRNA Detection Using an Oligonucleotide Labeled with PtCP
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
Photostability of StreptavidinPdCP, StreptavidinPtCP, DNAPtCP, and DNAPdCP Under In Situ Conditions
The photostability of PtCP and PdCP bound to streptavidin and DNA was investigated using slides stained for FISH. For streptavidinPtCP (F/P 5.6) or streptavidinPdCP (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 DNAPtCP/PdCP, the same probe was nick-translated using PtCP or PdCP-7dUTP. 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 biotin-based TSA was employed (
Embedding
All tissue sections stained with PtCP or PdCP conjugates were embedded in 25 µl 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 Netherlands). Tissue sections stained by immunoreagents conjugated to other fluorophores were embedded in 25 µl Vectashield (Vector; Burlingame, CA).
Immunocytochemical Staining of Glutathione Transferase
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-µm tissue sections were cut. The tissue sections were dried for 2 hr at 37C. To re-expose 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 anti-rabbit antibody 3 µg/ml (Vector); (b) TSA using biotintyramide; biotin deposits were detected with streptavidinPtCP (F/P 5.6) (5 µg/ml) or streptavidinPdCP (F/P 1.1 and 4.3), or streptavidinFITC (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, 2535 contrast values were obtained, averaged, and the standard deviation was calculated.
Immunohistochemical Detection of Prostate-specific Antigen (PSA)
Five-µm 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-rabbitFITC (4 µg/ml); (b) goat anti-rabbitPtCP (5µg/ml) (conjugates with different F/P ratios); (c) goat anti-rabbitPdCP (5g/ml) (conjugates with different F/P ratios); (d) HRP-labeled goat anti-rabbit after which the TSA system was applied (see above). The deposited biotin was detected with streptavidinPTCP (F/P 5.6) or streptavidinPdCP (F/P 4.3) or streptavidinFITC (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
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
Immunohistochemical Detection of Glucagon in Human Pancreas Tissue
Five-µm 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-rabbitPtCP or goat anti-rabbitPdCP 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 streptavidinPdCP (F/P 4.3), streptavidinPtCP (F/P 5.6), or streptavidinFITC, 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 (
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 500560-nm excitation filter, a 580-nm dichroic beam splitter, and a 600700-nm bandpass filter for selection of the emission. A x40 NA 1.30, a x100 1.30 PL Fluortar, or a x63 NA 1.32 PL APO objective was used, using 640 ASA 3M color slide film. The exposure times were 25 sec (DAPI counterstain) and 25 min (porphyrin).
Time-resolved Microscopy
Delayed imaging was accomplished using a special module incorporated into the DM microscope. It consists of a mini-chopper 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 allow 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 µs. The system suppresses fluorescence of shorter lifetimes by at least 1000-fold.
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Results |
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Photophysical Properties of PtCP and PdCP
The photophysical properties of free PtCP or of PtCPbound to streptavidin or antibodies have been determined previously (
Furthermore, the replacement of platinum by palladium into the porphyrin ring structure increases the lifetime by a factor of 10 to about 1000 µs (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 resulted in a significant reduction of F/P x Q value, which is an indication of the overall quantum yield of a particular conjugate. As an exception, no decrease in F/P x 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-7dUTP and PdCP-7dUTP
A threefold excess of PtCP-NHS ester was found optimal at 2-hr reaction time to convert AAdUTP into PtCP-7dUTP and PdCP-7dUTP (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 porphyrins did not change very much upon covalent conjugation to allylaminedUTP. 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 porphyrindUTPs strongly changed when Vectashield was used as anti-fading agent.
PtCP-7dUTP and PdCP-7dUTP 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-12dUTP 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 µM. In a standard nick translation, such a concentration will not be reached.
The nick translation was optimized by applying various dTTP/PtCPdUTP ratios into the nick-translation medium. The labeled probes were hybridized and the intensity of the spots was measured. Maximal intensity was achieved for 3090% of PtCPdUTP (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 allylaminedUTP) provided the brightest spots (approximately 1.7-fold compared to enzymatic labeling) (Figure 3). Similar results were obtained when this experiment was performed with fluoresceinNHS ester (data not shown). This increase in signal intensity is explained by a higher porphyrin or fluorescein density in the probe.
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Use of Streptavidin(Pt/Pd)CP, (Pt/Pd)CP-7dUTP for DNA In Situ Hybridization
Streptavidin(Pt/Pd)CP and (Pt/Pd)CP-7dUTP were evaluated for DNA in situ hybridization for the detection of DNA sequences of various sizes (Table 3). The 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 time-resolved microscope in the delayed mode.
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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 unlabeled oligo. A 50-fold excess of PtCP-2NHS resulted in a labeling percentage of at least 90%.
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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 Red-labeled 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.
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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 T1T1 and T1S0 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.
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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 islets, 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 not increase the staining intensity. No staining was observed when the primary anti-glucagon antibody was omitted from each particular detection method.
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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, PtCP- and 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 streptavidinPtCP conjugate with F/P 5.6 had the highest contrast value, e.g., 17.2, which is fourfold more than streptavidinFITC. For streptavidinPdCP, 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.
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Detection of p53 in Liver Tissue
Liver sections of N-OHAAF-treated rats showed positive staining for p53 (Figure 11) utilizing TSA (biotin) after the ABC method detected with fluorescein-labeled streptavidin. p53 was localized in the nucleus, as shown by DAPI counterstaining. Detection of TSA-deposited 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 Prostate-specific Antigen
The strong autofluorescence of the stroma results in a poor contrast for PSA staining using a fluorescein-labeled 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 orange-like. In this situation, the highest contrast was obtained using the time-resolved module (Figure 12). 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 streptavidinPdCP 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 detection, 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 anti-digoxygenin 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.
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Discussion |
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We recently reported on Pt-coproporphyrin-labeled antibodies and streptavidin for time-resolved microscopy (
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 glycerol/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 (
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 (
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-12dUTP, 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/PddUTPs. 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 allylaminedUTP (AA-dUTP) should be incorporated, which then can be labeled with the Pt/PdCPNHS 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 time-resolved 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.
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Acknowledgments |
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Supported by Technology Foundation (NWO, The Netherlands) grant LGN 22.2734 and in part by Boehringer Mannheim (Penzberg, Germany) and Leica (Wetzlar, Germany).
Received for publication January 27, 1998; accepted August 24, 1998.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams JC (1992) Biotin amplification of biotin and horseradish peroxidase signals. J Histochem Cytochem 40:1457-1463
Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ (1989) Catalyzed reporter deposition, a novel method of signal amplification: application to immunoassays. J Immunol Methods 125:279-285[Medline]
Canas LA, Busscher M, Angenebt GC, Beltran JP, van Tunen A (1994) Nuclear localization of the petunia MADS box protein FBP1. Plant J 6:597-604
Cooke HJ, Hindley J (1979) Cloning human satellite III DNA: different components are on different chromosomes. Nucleic Acids Res 6:3177-3197[Abstract]
de Haas RR, van Gijlswijk RPM, van de Tol EB, Zijlmans HMAA, BakkerSchut T, Bonnet J, Verwoerd NP, Tanke HJ (1997) Platinum porphyrins as phosphorescent label for time-resolved microscopy. J Histochem Cytochem 45:1279-1292
de Haas RR, Verwoerd NP, van der Corput MP, van Gijlswijk RPM, Siitari H, Tanke HJ (1996) The use of peroxidase-mediated deposition of biotintyramide in combination with time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization. J Histochem Cytochem 44:1091-1099
Florijn RJ, Slats J, Tanke HJ, Raap AK (1995) Analysis of antifading reagents for fluorescence microscopy. Cytometry 19:177-182[Medline]
Jovin TM, ArndtJovin DJ (1989) Luminescence digital imaging microscopy. Annu Rev Biophys Chem 18:217-308
Koes RE, Spelt CE, Mol JNM (1989) The chalcone synthase multigene family of Petunia hybrida (V30): differential, light-regulated expression during flower development and UV light induction. Plant Mol Biol 12:213-225
Landegent JE, Jansen in de Wal N, van Ommen GJB, Baas F, Vijlder JJM, van Duijn P, van der Ploeg (1985) Chromosomal localization of a unique gene by non-autoradiographic in situ hybridization. Nature 317:175-177[Medline]
Langer PR, Waldrop AA, Ward DC (1981) Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci USA 78:6633-6637[Abstract]
Marriott G, Clegg RM, Jovin DJ, Jovin TM (1991) Time-resolved imaging microscopy: phosphorescence and delayed fluorescence imaging. Biophys J 61:1374-1387
Marriott G, Heidecker M, Diamandis EP, YanMarriott Y (1994) Time-resolved delayed luminescence image microscopy using an europium ion chelate complex. Biophys J 67:957-965[Abstract]
Merz H, Malisius R, Mannweiler S, Zhou R, Hartmann W, Orschesschek K, Moubayed P, Feller AC (1995) ImmunoMax. An enhancement of hidden antigens. Lab Invest 73:149-156[Medline]
Raap AK, van de Corput MPC, Vervenne RAW, van Gijlswijk RPM, Tanke HJ, Wiegant J (1995) Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides. Hum Mol Genet 4:529-534[Abstract]
Ruizeveld de Winter JA, Trapman J, Brinkmann AO, Boersma WJA, Mulder E, Schroeder FH, Claassen E, van der Kwast ThH (1990) Androgen receptor heterogeneity in human prostatic carcinomas visualized by immunohistochemistry. J Pathol 161:329-332
Seveus L, Väisälä M, Syrjänen S, Sandberg M, Kuusisto A, Harju R, Salo J, Hemmilä I, Kojola H, Soini E (1992) Time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization. Cytometry 13:329-338[Medline]
Tanke HJ, Florijn RJ, Wiegant J, Raap AJ, Vrolijk J (1995) CCD microscopy and image analysis of cells and chromomsomes stained by fluorescence in situ hybridization. Histochem J 27:4-14[Medline]
van Blokland R, van der Geest N, Mol JNM, Kooter JM (1994) Transgene-mediated suppression of chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover. Plant J 6:861-877
van der Corput MPC, Dirks RW, van Gijlswijk RPM, FM van de Rijke, Raap AK (in press) Fluorescence in situ hybridization using horseradish peroxidase labeled oligodeoxynucleotides and tyramide signal amplification for sensitive DNA and mRNA detection. Histochem Cell Biol
van Gijssel HE, Maassen CBM, Mulder GJ, Meerman JHN (1997) p53 protein expression by hepatocarcinogens in the rat liver and its potential role in mitoinhibition of normal hepatocytes as a mechanism of hepatic tumour promotion. Carcinogenesis 18:1027-1033[Abstract]
van Gijssel HE, van Gijlswijk RPM, de Haas RR, Stark C, Mulder GJ, Meerman JHN (1998) Immunohistochemical visualisation of wild type p53 protein in paraffin-embedded rat liver using tyramide amplification: zonal hepatic distribution of p53 protein after N-hydroxy-2-acetylaminofluorene administration. Carcinogenesis 19:219-222[Abstract]
Verwoerd NP, Hennink EJ, Bonnet J, van der Geest CRG, Tanke HJ (1994) Use of ferro-electric liquid crystal shutters for time-resolved fluorescence microscopy. Cytometry 1:113-117
Waye JS, Willard HF (1986) Structure, organization and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome. Mol Cell Biol 6:3156-3165[Medline]
Waye JS, Willard (1989) Human beta satellite DNA: genomic organization and sequence definition of a class of highly repetitive tandem DNA. Proc Natl Acad Sci USA 86:6250-6254[Abstract]
Werner M, von Wasielewski R, Komminoth P (1996) Antigen retrieval, signal amplification and intensification in immunohistochemistry. Histochem Cell Biol 105:253-260[Medline]
Wiegant J, Ried T, Nederlof PM, van der Ploeg M, Tanke HJ, Raap AK (1991) In situ hybridization with fluoresceinated DNA. Nucleic Acids Res 19:3237-3241[Abstract]
Wiegant J, Wiesmijer CC, Hoovers JMN, Schuuring E, d'Azzo A, Vrolijk J, Tanke HJ, Raap AK (1993) Multiple and sensitive fluorescence in situ hybridization with rhodamine-, fluorescein-, and coumarin-labeled DNAs. Cytogenet Cell Genet 63:73-76[Medline]