TECHNICAL NOTE |
Correspondence to: Marlyse C. Knuchel, University Hospital, Div. of Infectious Diseases, Raemistr. 100, CH-8091 Zurich, Switzerland.
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Summary |
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We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 µg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH. (J Histochem Cytochem 48:285293, 2000)
Key Words: fluorescent in situ hybridization, ssDNA probes, RNA probes, HIV-1
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Introduction |
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Human immunodeficiency virus (HIV) infections can often be effectively suppressed with potent drug combinations, leading to undetectable plasma viral loads (
In situ hybridization (ISH) is useful for determining the phenotypes of HIV-bearing cells and for visualizing the infection in the context of surrounding tissue. However, disadvantages of ISH include the limited amount of cells or tissue that can be tested and the limited sensitivity of the method. The type of probe and the detection system are primary determinants of ISH sensitivity. One of the best HIV-1 ISH methods employs fragmented plasmid-derived RNA probes (
The most sensitive HIV-1 ISH detection systems to date have usually involved radioactively labeled probes (
We have developed a new method to generate single-stranded (ss) digoxigenin-labeled DNA probes with good yields and high specific activity. These probes were detected in situ using a peroxidaseanti-digoxigenin antibody followed by tyramide signal amplification to generate an exceptionally robust and sensitive fluorescent signal. This report describes the characterization of these methods using HIV-infected and uninfected cell lines.
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Materials and Methods |
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Cell Preparation
The 8E5 cell line harbors one HIV-1LAI provirus per cell (
Generation of Single-stranded (ss) DNA Probes
Stocks of dUTP-labeled PCR products were prepared by amplifying 20 ng of the pBH10 plasmid (AIDS Research and Reference Reagent Program; Rockville, MD) (
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An aliquot (150 ng) of each dUTP-labeled product was then used in an asymmetric PCR optimized to produce single-stranded, digoxigenin-labeled antisense products. Amplification reactions (100 µl) contained 3 µM (1.8 x 1014 molecules) of the lower primer, 1 x PCR buffer with 2.5 mM Mg (Perkin Elmer; Norwalk, CT), 0.2 mM dATP/dCTP/dGTP (Pharmacia Biotech; Piscataway, NJ), 0.05 mM dig-11-dUTP (Boehringer Mannheim), 0.15 mM dTTP (Pharmacia; Uppsala, Sweden), and 2.5 U AmpliTaq polymerase (Perkin Elmer). The cycling conditions were 35 cycles at 96C for 30 sec, 55C for 30 sec, 72C for 1 min, ending with 5 min at 72C. The seven asymmetrically amplified products were then precipitated (0.1 volume of 3 M Na-acetate + 2.5 volumes ethanol) and resuspended in 20 µl deionized water. The PCR reaction products, which still contained the input dUTP-dsDNA, were digested with uracilDNA glycosylase (0.7 U per 100 ng dUTP DNA) (New England BioLabs; Schwalbach, Germany), which cleaves dUTP but not dig-dUTP, according to the manufacturer's instructions. The reaction was stopped by incubation for 10 min at 95C and by adding 0.7 U uracil glycosylase inhibitor per 100 ng dUTP DNA (BioLabs). The resulting digoxigenin-labeled ssDNA probe (ssDNA) was purified with the QIAquick PCR purification kit (Qiagen; Hilden, Germany) and resuspended in TE buffer. The final product was controlled for size and purity by electrophoresis on 6% nondenaturing polyacrylamide gels or 2% agarose gels, in which each incorporated dig-11-dUTP retards migration as if it were two unlabeled nucleotides (
Generation of RNA Probes
Single-stranded, digoxigenin-labeled RNA probes (PCR-RNA) were generated using a two-step PCR amplification method followed by in vitro RNA transcription (
The RNA products (20 µl) were next incubated with 2 U DNase I (Boehringer Mannheim), precipitated with 1/10 volume 4 M LiCl/3 volumes EtOH, and resuspended in DEPC water. To determine specific activity, purified RNA products were electrophoresed in a 1.5% agarose formaldehyde gel, transferred to a nylon membrane, and bound digoxigenin was detected using anti-digoxigenin alkaline phosphatase followed by NBT/BCIP precipitation (all Boehringer). The blots were digitally imaged and bands were quantified using image analysis software (Intelligent Quantifier).
Plasmid-derived RNA Probe
Digoxigenin-labeled RNA probes (Lofstrand Labs; Gaithersburg, MD) were synthesized by in vitro transcription of plasmids F (2.7 kb), H (1.4 kb), I (2.6 kb), and J (1.1 kb) and carbonate-sheared to 350 nts average length (
In Situ Hybridization and Signal Amplification
Frozen slides with dried cells were thawed, fixed in 4% paraformaldehyde for 10 min, washed in PBS, and incubated for 10 min at 37C in 1 µg/ml proteinase K. Slides with H9 cells were used throughout as controls and were hybridized with the same procedures as used for the 8E5 cells. The cells were then washed and acetylated for 15 min using 0.1 M TEA with two additions of acetanhydride. The slides were then rinsed in PBS and 2 x SSC, dehydrated in graded ethanols, and air-dried. Hybridization solution (20 µl final volume containing 4 x SSC, 2 x Denhardt's solution, 600 µg/ml herring testis DNA, 45% formamide, 1 U/µl RNasin, 5 mM DTT) including 1200 ng probe mix was applied to each half of a slide, coverslipped, and incubated overnight at 50C. After hybridization, the slides were washed for 5 min at 45C in 2 x SSC with 45% formamide, 15 min at 45C in 2 x SSC, and 10 min at room temperature (RT) in 2 x SSC. Nonspecifically bound RNA probes were digested with RNase (12 mg/ml for 30 min) and the slides were washed in 2 x SSC again for 5 min at 37C, 10 min at 45C, and 5 min at RT. Endogenous peroxidases were inactivated using PBS containing 3% H2O2 for 10 min before rinsing the slides in TNT (150 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5).
To detect the digoxigenin-labeled probes, slides were first blocked for 30 min in TNB [150 mM Tris, 150 mM NaCl, 0.5% blocking reagent (NEN Life Science; Boston, MA)] and an anti-digoxigeninperoxidase antibody (Boehringer Mannheim) diluted 1:250 in TNB was then applied for 30 min. Amplification of the signal was achieved using the TSA-direct Cyanine 3 FISH kit (NEN), whereby the signal was developed by an incubation of 1:50 Cy3 tyramide in amplification buffer for 30 min. The cells were finally counterstained with 0.5 µM DAPI (Sigma; Buchs, Switzerland), washed, dehydrated, and mounted using ProLong Antifade kit (Molecular Probes).
Quantitation of Fluorescent ISH
Fluorescence was detected using a DM/RXA microscope (Leica; Wetzlar, Germany) fitted with a black-and-white cooled CCD digital camera (Micromax RTE/CCD 1317-K1; Princeton Instruments, Trenton, NJ) connected to an Intel 586-based desktop computer running MetaMorph Image Analysis software (version 3.5; Universal Imaging, West Chester, PA) in a Windows 95 environment. Using a Plan Fluotar 40x/0.7 objective (Leica), each microscopic field was imaged twice, applying a different filter set each time, to independently detect Cy3 and DAPI (filter sets HQ 41007a and TR-1, respectively; Chroma, Brattleboro, VT). The DAPI staining was used to visualize nuclei. The dark current (between-cell) fluorescence was subtracted from the respective images before a mask was generated from the Cy3 fluorescence. The creation of a mask permitted the measurement of individual cells and defined the fluorescent intensity of the signal in each cell, which is expressed as gray value units (GV) in the image analysis software. Because the signals on H9 cells were very low, the exposure time to generate an image was increased from 100 msec, used for 8E5 cells, to 1 sec. For comparisons among images, GVs from 1-sec exposures were divided by 10 to derive equivalent 100 msec GVs. This correction was empirically validated in separate experiments. All images were similarly scaled so that intensities among different images could be directly compared.
Statistics
The capacity of each probe to differentiate between true positive and negative values was evaluated. Using mean and standard deviation of GVs of uninfected H9 cells, we calculated for each time point the probability (cumulative density of the normal distribution) of the lowest measured GV value of infected 8E5 cells to be falsely classified as negative.
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Results |
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ssDNA Probe Synthesis
In efforts to improve the ISH method, we tested whether ssDNA probes would result in increased sensitivity as compared to PCR-amplified, T7-transcribed RNA probes (PCRRNA). For this, we generated digoxigenin-labeled single-stranded RNA (PCR-RNA) and digoxigenin-labeled ssDNA probes (Figure 1). The mix of 22 PCR-RNA probes covered 63% of the HIV-1 genome and had an average length of 294 nts (range 204301 nts) (Table 1). Seven ssDNA probes averaging 725 nts in length (range 641923 nts) and covering 55% of the genome were also synthesized (Table 1). Each synthesis resulted in 0.51 µg purified ssDNA probe. To ensure that the generated products were truly single stranded and of the expected size, double-stranded as well as single-stranded products were analyzed by gel electrophoresis (Figure 2). The purified ssDNA probes migrated more slowly than predicted by size alone because of digoxigenin incorporation. Similar results were obtained using 6% nondenaturing polyacrylamide gels followed by membrane transfer and digoxigenin detection.
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Maximal yields and purity of the probes were achieved by optimizing the purification steps. To prevent incorporation of dUTP and digestion of the ssDNA probes by uracilDNA glycosylase, dUTP from the first amplification had to be eliminated before the second asymmetric PCR. The dUTP-labeled products were therefore purified through a DNA purification column followed by NaAc/isopropanol precipitation. Two purification columns (Boehringer Mannheim and Qiagen) were evaluated for yields and purity of the product. In preliminary experiments, shorter ssDNA probes of 200300 nts were synthesized but neither purification column produced adequate or reproducible ssDNA recovery (not shown). By increasing the size of the probes to 641923 nts, ssDNA yields increased to 8090% for both columns. The second purification step, the NaAc/isopropanol precipitation, was chosen because isopropanol preferentially precipitates longer DNA pieces. We anticipated that unwanted dUTP would thereby be eliminated. NaAc/isopropanol precipitation resulted in 5075% recovery, depending on the size of the ssDNA pieces, and the final product was free of primers, as documented by gel electrophoresis. After asymmetric PCR amplification, it was necessary to precipitate the DNA to change buffer for the enzymatic digestion with uracilDNA glycosylase. To this end, the most efficient precipitation method was chosen (NaAc/ethanol). The DNA recovery of this method reaches almost 100%. However, it also precipitates primers and other short DNA pieces. After digestion, the digoxigenin-labeled ssDNA probes were separated from primers and digested DNA pieces using the QIAquick PCR purification kit, resulting in high yields of purified ssDNA probes.
Hybridization Characteristics of ssRNA and ssDNA Probes
The 8E5 cell line, which constitutively expresses HIV-1, and the uninfected H9 cells were used to test ISH with the digoxigenin-labeled ssDNA, PCR-RNA, and plasmidRNA probes (Figure 3 Figure 4 Figure 5). Each probe was tested at concentrations of 1, 5, 20, 50, 100, and 200 ng per hybridization. Hybridized probes were visualized using Cy3 TSA amplification and imaged in two colors; DAPI and Cy3. An average of 200 representative cells (range 160253) from each hybridization was analyzed using image analysis software.
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The fluorescent intensity (gray value units, GV) differences among the probe mixes were evident when hybridized 8E5 and H9 cells were viewed. A representative example of hybridization of these cells with 100 ng of probe mix is shown in Figure 3. Infected cells hybridized with ssDNA were frequently filled with strong Cy3 fluorescence (Figure 3A and Figure 3B), whereas the RNA probe mixes typically produced smaller areas of Cy3 positivity (Figure 3D, Figure 3E, Figure 3G, and Figure 3H). Furthermore, the ssDNA probe produced higher fluorescent intensities than either RNA probe (compare Figure 3B with Figure 3E and Figure 3H). The uninfected H9 cell controls showed low levels of nonspecific Cy3 fluorescence that could be differentiated from the stronger, true-positive signals by visual examination and by digital image analysis (Figure 3C, Figure 3F, and Figure 3I).
Titrations of the probe mixes revealed that all three probe types reached maximal signal intensities with 20 ng probe per hybridization (Figure 4). At very low concentrations, such as 5 ng/hybridization, the ssDNA probe did not differentiate infected from uninfected cells as effectively as the PCR-RNA and plasmidRNA probes. However, when 20 ng or more probe was used, no overlaps between 8E5 GV/cell and H9 GV/cell were observed for any of the probe types in these experiments. The ssDNA probe produced brighter signals overall. The 8E5 GV/cell medians with 20 ng probe were 3.03 x 106 (ssDNA), 5.48 x 105 (PCR-RNA), and 6.92 x 105 (plasmidRNA). The sensitivity of the probes was also demonstrated by analyzing digital images to define the percentage of 8E5 cells with measurable HIV-1 RNA by ISH. Starting with 20 ng, all of the probes recognized 100% of the 8E5 cells as being HIV-1-positive (Figure 4). Nonspecific fluorescence from hybridized H9 cells was less dependent on probe concentration, ranging from 8565 to 19,850 median GV/cell across all probe concentrations and probe types (Figure 4).
Differentiation of negative from weakly positive cells was substantially more robust with the ssDNA probe. This is indicated by the separation between the minimal true-positive 8E5 cell intensity and the maximal true-negative H9 cell intensity, as shown in Figure 4. The power of distinguishing true-negative from true-positive cells was quantified by calculating the probability that the lowest intensity true-positive cell is a member of the set of true-negative intensity values for each probe type and probe concentration (Figure 5). None of the probe types effectively separated low-positive 8E5 cells from the normal distribution of true-negative H9 cells when only 1 ng probe/hybridization was used (p>0.09 for all three probe types). However, with 20 ng/hybridization or more, the ssDNA probe was three to four orders of magnitude more effective at minimizing the probability of overlap between the lowest positive and true-negative cell intensities (Figure 5).
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Discussion |
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This report describes a novel, convenient method for generating single-stranded, digoxigenin-labeled DNA probes. This method is easy to perform, reproducible, and generates up to 1 µg ssDNA per amplification. ISH with ssDNA probes resulted in strong fluorescent intensities and excellent discrimination between infected and uninfected cells. These ssDNA probes demonstrated better sensitivity and specificity than equivalent RNA probes derived from plasmids or PCR products at probe concentrations >20 ng/hybridization.
Previous reports of ssDNA probes demonstrated a trend for these probes to be more sensitive than denatured dsDNA probes (
The method reported here for synthesis of digoxigenin-labeled ssDNA is, to our knowledge, the only one that eliminates any double-stranded input DNA, resulting in a high purity of ssDNA probes. Although we have used these probes only for ISH, they may also be useful for other techniques involving hybrid detection. Major advantages of this ssDNA synthesis method include streamlined laboratory methods, high yields of up to 1 µg per synthesis, generation of well-defined probes without subcloning, and relatively low cost per hybridization. In addition, the ssDNA synthesis protocol ensures relatively high purity and specific activity, which may contribute to the improved performance of these probes.
Long ssDNA probes offer theoretical and practical advantages compared with probes composed of dsDNA, ssRNA, or oligonucleotides. The efficacy of denatured double-stranded probes suffers from hybrid competition between the complementary probe strand and the target, which is eliminated with single-stranded probes (
A possible explanation for better performance of the ssDNA probe mix could relate to probe length, because the average length of the ssDNA probes (725 nts) was greater than the PCR-RNA (294 nts) and plasmidRNA (350 nts) probes. We therefore compared 24 ssDNA probes with a mean length of 286 nts covering 66% of the HIV-1 genome with our seven ssDNA probe mix and hybridized 8E5 and H9 cells, respectively. These probes generated results similar to those achieved with seven long ssDNA probes covering 55% of the genome, and no differences in dynamics or hybridization level were observed (not shown). Similar observations have been made previously, comparing probes between 151 nts and 749 nts (
All three probe types tested were capable of separating all true-positive 8E5 cells from all true-negative H9 cells when at least 20 ng probe/hybridization was used. However, the ssDNA probe produced larger separations between negative and positive cells, effectively reducing the chance of misclassifying low-positive cells as HIV-negative. Because 8E5 cell cultures include individual cells with very low and very high HIV-1 RNA expression (unpublished data), weakly positive 8E5 cells represent low-level expression, a condition that might be encountered in vivo. We previously reported that radioactively-labeled, PCR-derived RNA probes covering 20% of the HIV-1 genome produced brighter signals and less background than plasmid-derived RNA probes covering 34% of the genome (
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Acknowledgments |
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Supported by grants from the Swiss National Science Foundation (32-43654) and the EMDO Stiftung Zurich.
Received for publication June 24, 1999; accepted August 31, 1999.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akam ME (1983) The location of Ultrabithorax transcripts in Drosophila tissue sections. EMBO J 2:2075-2084[Medline]
An SF, Franklin D, Fleming KA (1992) Generation of digoxigenin-labelled double-stranded and single-stranded probes using the polymerase chain reaction. Mol Cell Probes 6:193-200[Medline]
Berger CN (1986) In situ hybridization of immunoglobulin-specific RNA in single cells of the B lymphocyte lineage with radiolabelled DNA probes. EMBO J 5:85-93[Abstract]
Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ (1989) Catalized reporter deposition, a novel method of signal amplification. Application to immunoassays. J Immunol Methods 125:279-285[Medline]
Bobrow MN, Shaughnessy KJ, Litt GJ (1991) Catalyzed reporter deposition, a novel method of signal amplification. II. Application to membrane immunoassays. J Immunol Methods 137:103-112[Medline]
Celeda D, Bettag U, Cremer C (1992) PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization. Biotechniques 12:98-102[Medline]
Collier AC, Coombs RW, Schoenfeld DA, Bassett R, Baruch A, Corey L (1996) Combination therapy with zidovudine, didanosine and saquinavir. Antiviral Res 29:99[Medline]
Cone RW, Schlaepfer E (1997) Improved in situ hybridization to HIV with RNA probes derived from PCR products. J Histochem Cytochem 45:721-727
Cox KH, DeLeon DV, Angerer LM, Angerer RC (1984) Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol 101:485-502[Medline]
Dallman MJ, Montgomery RA, Larsen CP, Wanders A, Wells AF (1991) Cytokine gene expression: analysis using northern blotting, polymerase chain reaction and in situ hybridization. Immunol Rev 119:163-179[Medline]
De Jong AS, Van Kesselvan Vark M, Raap AK (1985) Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry. Histochem J 17:1119-1130[Medline]
Finzi D, Hermankova M, Pierson T, Carruth LM, Buck C, Chaisson RE, Quinn TC, Chadwick K, Margolick J, Brookmeyer R, Gallant J, Markowitz M, Ho DD, Richman DD, Siliciano RF (1997) Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278:1295-1300
Folks TM, Powell D, Lightfoote M, Koenig S, Fauci AS, Benn S, Rabson A, Daugherty D, Gendelman HE, Hoggan MD, Venkatesan S, Martin MA (1986) Biological and biochemical characterization of a cloned Leu-3-cell surviving infection with the acquired immune deficiency syndrome retrovirus. J Exp Med 164:280-290[Abstract]
Haase AT, Henry K, Zupancic M, Sedgewick G, Faust RA, Melroe H, Cavert W, Gebhard K, Staskus K, Zhang ZQ, Dailey PJ, Balfour HHJ, Erice A, Perelson AS (1996) Quantitative image analysis of HIV-1 infection in lymphoid tissue. Science 274:985-989
Hahn BH, Shaw GM, Arya SK, Popovic M, Gallo RC, Wong SF (1984) Molecular cloning and characterization of the HTLV-III virus associated with AIDS. Nature 312:166-169[Medline]
Hannon K, Johnstone E, Craft LS, Little SP, Smith CK, Heiman ML, Santerre RF (1993) Synthesis of PCR-derived, single-stranded DNA probes suitable for in situ hybridization. Anal Biochem 212:421-427[Medline]
Hunyady B, Krempels K, Harta G, Mezey E (1996) Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining. J Histochem Cytochem 44:1353-1362[Abstract]
Klein SC, Golverdingen JG, Bouwens AG, Tilanus MG, de Weger RA (1995) An alternatively spliced interleukin 4 form in lymphoid cells. Immunogenetics 41:57[Medline]
Michel D, Trembleau A, Moyse E, Brun G (1997) Optimization of PCR/lambda exonuclease-mediated synthesis of sense and antisense DNA probes for in situ hybridization. Histochem J 29:685-693[Medline]
Petrusz P, Ordronneau P, Finley JC (1980) Criteria of reliability for light microscopic immunocytochemical staining. Histochem J 12:333-348[Medline]
Popovic M, Sarngadharan MG, Read E, Gallo RC (1984) Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224:497-500[Medline]
Rigby PW, Dieckmann M, Rhodes C, Berg P (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237-251[Medline]
Romano GJ, Krust A, Pfaff DW (1989) Expression and estrogen regulation of progesterone receptor mRNA in neurons of the mediobasal hypothalamus: an in situ hybridization study. Mol Endocrinol 3:1295-1300[Abstract]
Scopsi L, Larsson LI (1986) Increased sensitivity in peroxidase immunocytochemistry. A comparative study of a number of peroxidase visualization methods employing a model system. Histochemistry 84:221-230[Medline]
Straus W (1982) Imidazole increases the sensitivity of the cytochemical reaction for peroxidase with diaminobenzidine at a neutral pH. J Histochem Cytochem 30:491-493[Medline]
Van Heusden J, de Jong P, Ramaekers F, Bruwiere H, Borgers M, Smets G (1997) Fluorescein-labeled tyramide strongly enhances the detection of low bromodeoxyuridine incorporation levels. J Histochem Cytochem 45:315-319
Wong JK, Gunthard HF, Havlir DV, Zhang ZQ, Haase AT, Ignacio CC, Kwok S, Emini E, Richman DD (1997a) Reduction of HIV-1 in blood and lymph nodes following potent antiretroviral therapy and the virologic correlates of treatment failure. Proc Natl Acad Sci USA 94:12574-12579
Wong JK, Hezareh M, Gunthard HF, Havlir DV, Ignacio CC, Spina CA, Richman DD (1997b) Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 278:1291-1295