In Situ Hybridization AT-Tailing with Catalyzed Signal Amplification for Sensitive and Specific in Situ Detection of Human Immunodeficiency Virus-1 mRNA in Formalin-Fixed and Paraffin-Embedded Tissues
2003; Elsevier BV; Volume: 162; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)63833-3
ISSN1525-2191
AutoresNoriko Nakajima, Petronela Ionescu, Yuko Sato, Michie Hashimoto, Toshihiro Kuroita, Hidehiro Takahashi, Hiroshi Yoshikura, Tetsutaro Sata,
Tópico(s)RNA Interference and Gene Delivery
ResumoIn situ hybridization is one of the most important techniques to visualize gene expression at the cellular level in various tissues. The in situ hybridization-AT tailing (ISH-AT) method uses a specially designed and synthesized oligonucleotide probe that has (AT)10 on the 3′ side. This (AT)10 of the probe is elongated by ΔTth DNA polymerase in the presence of dATP, dTTP, and labeled dUTP in the tissue after hybridization. Through this process the target is labeled with many hapten molecules. In this study, we detected human immunodeficiency virus type 1 RNA in formalin-fixed and paraffin-embedded tissues obtained from autopsied patients with acquired immunodeficiency syndrome by combining ISH-AT with the catalyzed signal amplification (CSA) system (ISH-AT-CSA), although we failed to detect signals from the same samples by conventional in situ hybridization using RNA probes (RISH) with CSA (RISH-CSA). We demonstrated that the ISH-AT-CSA method was superior to RISH-CSA in terms of both sensitivity and specificity, and that it was applicable to fluorescence in situ hybridization and double staining with immunohistochemistry for the characterization of cell phenotypes. In situ hybridization is one of the most important techniques to visualize gene expression at the cellular level in various tissues. The in situ hybridization-AT tailing (ISH-AT) method uses a specially designed and synthesized oligonucleotide probe that has (AT)10 on the 3′ side. This (AT)10 of the probe is elongated by ΔTth DNA polymerase in the presence of dATP, dTTP, and labeled dUTP in the tissue after hybridization. Through this process the target is labeled with many hapten molecules. In this study, we detected human immunodeficiency virus type 1 RNA in formalin-fixed and paraffin-embedded tissues obtained from autopsied patients with acquired immunodeficiency syndrome by combining ISH-AT with the catalyzed signal amplification (CSA) system (ISH-AT-CSA), although we failed to detect signals from the same samples by conventional in situ hybridization using RNA probes (RISH) with CSA (RISH-CSA). We demonstrated that the ISH-AT-CSA method was superior to RISH-CSA in terms of both sensitivity and specificity, and that it was applicable to fluorescence in situ hybridization and double staining with immunohistochemistry for the characterization of cell phenotypes. In the postgenomic era, it will be crucial to image both gene and protein expressions at the cellular level in a variety of tissues. In situ hybridization and immunohistochemistry are to be the important techniques to achieve this. Several methods of in situ hybridization have been developed, and its sensitivity and specificity of the signal-to-noise (S/N) ratio, preservation of tissue morphology, and preparation of probes have been improved.1Corput MPC Dirks RW Gijlswijk PM Binnendijk E Hattinger CM Paus RA Landegent JE Raap AK Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification.J Histochem Cytochem. 1998; 46: 1249-1259Crossref PubMed Scopus (56) Google Scholar, 2Speel EJM Saremaslani P Roth J Hopman AHN Komminoth P Improved mRNA in situ hybridization on formaldehyde-fixed and paraffin-embedded tissue using signal amplification with different haptenized tyramides.Histochem Cell Biol. 1998; 110: 571-577Crossref PubMed Scopus (46) Google Scholar, 3Egger D Bolton R Rahner C Bienz K Fluorochrome-labeled RNA as a sensitive, strand-specific probe for direct fluorescence in situ hybridization.Histochem Cell Biol. 1999; 111: 319-324Crossref PubMed Scopus (27) Google Scholar, 4Speel EJM Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors.Histochem Cell Biol. 1999; 112: 89-113Crossref PubMed Scopus (52) Google Scholar, 5Luehrsen KR Davidson S Lee YJ Rouhani R Soleimani A Raich T Cain CA Collarini EJ Yamanishi DT Pearson J Magee K Madlansacay MR Bodepudi V Davoudzadeh D Schueler PA Mahoney W High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues.J Histochem Cytochem. 2000; 48: 133-145PubMed Google Scholar, 6Murakami T Hagiwara T Yamamoto K Hattori J Kasami M Utsumi M Kaneda T A novel method for detecting HIV-1 by non-radioactive in situ hybridization: application of a peptide nucleic acid probe and catalyzed signal amplification.J Pathol. 2001; 194: 130-135Crossref PubMed Scopus (25) Google Scholar, 7Qian X Bauer RA Xu HS Lloyd RV In situ hybridization detection of calcitonin mRNA in routinely fixed, paraffin-embedded tissue sections: a comparison of different types of probes combined with tyramide signal amplification.Appl Immunohistochem Mol Morphol. 2001; 9: 61-69Crossref PubMed Scopus (24) Google Scholar RNA probes have generally been used as the most sensitive probe and are considered suitable for the detection of very low amounts of transcripts. Compared to oligonucleotide probes, however, their specificity, signal-to-noise (S/N) ratio, and penetration into cells and tissues are inferior and probe preparation is more complicated. The use of small-sized probes such as oligonucleotide probes reduces noise signals originating from nonspecifically bound probes. Moreover, it is noted that we are able to prepare the oligonucleotide probe without the cloning steps for construction of plasmid DNAs. We developed a new type of probe, hybridization AT tailing (HybrAT) probe for filter hybridization, and a new in situ hybridization technique, ISH-AT, as described previously.8Nakajima N Hanaki K Shimizu YK Ohnishi S Gunji T Nakajima A Nozaki C Mizuno K Odawara T Yoshikura H Hybridization-AT-tailing (HybrAT) method for sensitive and strand-specific detection of DNA and RNA.Biochem Biophys Res Commun. 1998; 248: 613-620Crossref PubMed Scopus (6) Google Scholar, 9Nakajima N Sata T Hanaki K Kurata K Yoshikura H Application of hybridization AT-tailing method for detection of human immunodeficiency virus RNA in cells and simian immunodeficiency virus RNA in formalin-fixed and paraffin-embedded tissues.J Virol Methods. 1999; 81: 169-177Crossref PubMed Scopus (5) Google Scholar A HybrAT probe is an oligonucleotide probe consisting of a hybridization region and an AT tailing region, (AT)10, on the 3′ end. It should be noted that the probe was not labeled beforehand; however, after hybridization by incubation at a constant temperature (60°C) with a labeling solution consisting of dATP, dTTP, labeled dUTP (eg, biotin-16-dUTP, or digoxigenin-11-dUTP), and ΔTth DNA polymerase (Toyobo, Osaka, Japan), which mediated a rapid elongation of oligo-(AT)10 into high-molecular weight (AT)x copolymers with incorporation of labeled dUTP.10Hanaki K Odawara T Nakajima N Shimizu YK Nozaki C Mizuno K Muramatsu T Kuchino Y Yoshikura H Two different reactions involved in the primer/template independent polymerization of dATP and dTTP by Taq DNA polymerase.Biochem Biophys Res Commun. 1998; 244: 210-219Crossref PubMed Scopus (25) Google Scholar Furthermore, ISH-AT allowed detection of simian immunodeficiency virus (SIV) RNA in formalin-fixed and paraffin-embedded tissue sections of an experimentally infected monkey.9Nakajima N Sata T Hanaki K Kurata K Yoshikura H Application of hybridization AT-tailing method for detection of human immunodeficiency virus RNA in cells and simian immunodeficiency virus RNA in formalin-fixed and paraffin-embedded tissues.J Virol Methods. 1999; 81: 169-177Crossref PubMed Scopus (5) Google Scholar The sensitivity of this technique was at least comparable to that of in situ hybridization using RNA probes (RISH).9Nakajima N Sata T Hanaki K Kurata K Yoshikura H Application of hybridization AT-tailing method for detection of human immunodeficiency virus RNA in cells and simian immunodeficiency virus RNA in formalin-fixed and paraffin-embedded tissues.J Virol Methods. 1999; 81: 169-177Crossref PubMed Scopus (5) Google Scholar Recently, catalyzed signal amplification (CSA) or tyramide signal amplification systems have been introduced to amplify the signal detection by immunohistochemistry and in situ hybridization.1Corput MPC Dirks RW Gijlswijk PM Binnendijk E Hattinger CM Paus RA Landegent JE Raap AK Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification.J Histochem Cytochem. 1998; 46: 1249-1259Crossref PubMed Scopus (56) Google Scholar, 2Speel EJM Saremaslani P Roth J Hopman AHN Komminoth P Improved mRNA in situ hybridization on formaldehyde-fixed and paraffin-embedded tissue using signal amplification with different haptenized tyramides.Histochem Cell Biol. 1998; 110: 571-577Crossref PubMed Scopus (46) Google Scholar, 4Speel EJM Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors.Histochem Cell Biol. 1999; 112: 89-113Crossref PubMed Scopus (52) Google Scholar, 7Qian X Bauer RA Xu HS Lloyd RV In situ hybridization detection of calcitonin mRNA in routinely fixed, paraffin-embedded tissue sections: a comparison of different types of probes combined with tyramide signal amplification.Appl Immunohistochem Mol Morphol. 2001; 9: 61-69Crossref PubMed Scopus (24) Google Scholar, 11Bobrow MN Harris TD Shaughnessy KJ Litt GJ Catalyzed reporter deposition, a novel method of signal amplification.J Immunol Methods. 1989; 125: 279-285Crossref PubMed Scopus (644) Google Scholar, 12Van Gijlswijk RPM Zijlmans HJMAA Wiegant J Bobrow MN Erickson TJ Adler KE Tanke HJ Raap AK Fluorochrome-labeled tyramides: use in immunohistochemistry and fluorescence in situ hybridization.J Histochem Cytochem. 1997; 45: 375-382Crossref PubMed Scopus (165) Google Scholar, 13Schmidt BF Chao J Zhu Z DeBiasio RL Fischer G Signal amplification in the detection of single-copy DNA and RNA by enzyme-catalyzed deposition (CARD) of the novel fluorescent reporter substrate Cy3.29-tyramide.J Histochem Cytochem. 1997; 45: 365-373Crossref PubMed Scopus (50) Google Scholar, 14Speel EJM Hopman AHN Komminoth P Amplification methods to increase the sensitivity of in situ hybridization: play CARD(S).J Histochem Cytochem. 1999; 47: 281-288Crossref PubMed Scopus (156) Google Scholar, 15Strappe PM Wang TH McKenzie CA Lowrie S Simmonds P Bell JE Enhancement of immunohistochemical detection of HIV-1 p24 antigen in brain by tyramide signal amplification.J Virol Methods. 1997; 67: 103-112Crossref PubMed Scopus (19) Google Scholar, 16Wang G Achim CL Hamilton RL Wiley CA Soontornniyomkij V Tyramide signal amplification methods in multiple-label immunofluorescence confocal microscopy.Methods. 1999; 18: 459-464Crossref PubMed Scopus (68) Google Scholar, 17Yang H Wanner IB Roper SD Chaudhari N An optimized method for in situ hybridization with signal amplification that allows the detection of rare mRNAs.J Histochem Cytochem. 1999; 47: 431-445Crossref PubMed Scopus (163) Google Scholar In this study, we attempted to combine ISH-AT with CSA and succeeded in detecting HIV-1 RNA with a high signal-to-noise (S/N) ratio in sections of formalin-fixed and paraffin-embedded autopsy tissues. We failed to detect any signals in the same samples by combining a conventional RISH with CSA (RISH-CSA). This ISH-AT-CSA allowed detection of the signals using both chromogen for conventional histopathology and fluorescence for confocal laser microscopy to characterize the phenotype of the cells. The formalin-fixed and paraffin-embedded autopsy tissues selected in this study are summarized in Table 1. M1, a rhesus monkey (Macaca mulatta) inoculated intravenously with 10 TCID50 of SHIV89.6PD (the gag, pol, nef regions of the gene are derived from SIV), showed CD4 + T cell depletion and high-plasma viral load, and was autopsied 8 months after infection.18Matano T Kano M Nakamura H Takeda A Nagai Y Rapid appearance of secondary immune response and protection from acute CD4 depletion after highly pathogenic immunodeficiency virus challenge in macaques vaccinated with a DNA prime/Sendai virus vector boost regimen.J Virol. 2001; 75: 11891-11896Crossref PubMed Scopus (88) Google Scholar M2 was inoculated with SIVmac32H19Rud EW Cranage M Yon J Quirk J Ogilvie L Cook N Webster S Dennis M Clarke BE Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants.J Gen Virol. 1994; 75: 529-543Crossref PubMed Scopus (130) Google Scholar and was autopsied 1 year and 3 months after infection. They were serologically confirmed to be negative for SIV, simian type-D retrovirus, and simian T-cell leukemia virus type I before use and maintained in accordance with the institutional guidelines for laboratory animals. The lymph nodes were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and brains were fixed in 10% buffered formalin.Table 1Summary of Samples and Results of Immunohistochemistry, ISH-AT-CSA, and RISH-CSACaseTissueVirusAge*Time after inoculation in cases of monkey. (sex)SIVp27/HIVp24ISH-AT-CSARISH-CSAMonkey M1LNSHIV89.6PD†GenBank; U89134.8 months+++++Brain++++++ M2BrainSIVmac32H‡GenBank; D01065.1 year 3 months++++++Human H1LNHIV-16 months (M)+++− H2LNHIV-16 months (M)−++− H3LNHIV-17 months (M)++− H4LNHIV-19 months (M)++− H5LNHIV-11 year (M)−+− H6LNHIV-11 year (F)++− H7LNHIV-11 year (M)++++− H8LNHIV-11 year 1 month (F)++− H9LNHIV-11 year 2 months (M)++++− H10LNHIV-11 year 4 months (M)++− H11LNHIV-11 year 9 months (M)+++− H12LNHIV-12 years 1 month (F)++− H13LNHIV-12 years 4 months (F)++− H14LNHIV-17 years 4 months (M)+−ND H15LNHIV-110 years (M)−−ND H16BrainHIV-19 years (M)+++− H17BrainHIV-133 years (M)++++− H18BrainHIV-142 years (M)++−LN, lymph node; ND, not done.The signal intensity is presented as +++, strong; ++, moderate; +, mild; −, no signal.* Time after inoculation in cases of monkey.† GenBank; U89134.‡ GenBank; D01065. Open table in a new tab LN, lymph node; ND, not done. The signal intensity is presented as +++, strong; ++, moderate; +, mild; −, no signal. For detection of HIV-1 RNA, paraffin blocks of lymph node specimens (H1 to H15) were obtained from Romanian children who had died of pediatric acquired immunodeficiency syndrome (AIDS)-related illnesses. The brains (H16 to H18) were obtained from a Romanian child and Japanese adults who died of HIV-1 encephalitis. The HybrAT probe consists of two regions, hybridization and elongation regions (Figure 1). The hybridization region, a 40- to 50-base-long complementary sequence, is located on the 5′ side, and the AT tailing region for elongation, consisting of 10 repeats of AT, on the 3′ side.8Nakajima N Hanaki K Shimizu YK Ohnishi S Gunji T Nakajima A Nozaki C Mizuno K Odawara T Yoshikura H Hybridization-AT-tailing (HybrAT) method for sensitive and strand-specific detection of DNA and RNA.Biochem Biophys Res Commun. 1998; 248: 613-620Crossref PubMed Scopus (6) Google Scholar, 9Nakajima N Sata T Hanaki K Kurata K Yoshikura H Application of hybridization AT-tailing method for detection of human immunodeficiency virus RNA in cells and simian immunodeficiency virus RNA in formalin-fixed and paraffin-embedded tissues.J Virol Methods. 1999; 81: 169-177Crossref PubMed Scopus (5) Google Scholar The predicted Tm value of the hybridization sequence should be higher than the temperature for the elongation reaction (60°C) so that the probe will firmly bind to the target sequence during the elongation process. The sequences and positions of all oligonucleotides used in this study are presented in Table 2. Each sequence of the hybridization region was checked for absence of cross homologies with human and simian sequences. One biotin molecule was conjugated on the 5′ side of all probes for control experiments. The GC% of the hybridization region was 50 to 60%. All of the HybrAT probes were synthesized at Nihon Gene Research Laboratories, Inc. (Sendai, Japan) on order.Table 2Synthetic Oligonucleotides Used for Hybridization AT Tailing (HybrAT) ProbesProbePositionSequenceAS-5′ biotin-SIVnef(AT)109380–9419*Nucleotide positions refer to the SIVmac239 strain20 (GeneBank, M33262).5′ biotin-CCCATAAGTCTCCCCACGCGCCCGCAAGAGTCTCTGTCGCATATATATATATATATATAT-3′S-5′ biotin-SIVnef(AT)109419–93805′ biotin-GCGACAGAGACTCTTGCGGGCGCGTGGGGAGACTTATGGGATATATATATATATATATAT-3′AS-5′ biotin-HIVnef(AT)108910–8871†Nucleotide positions refer to pNL4-321 (GeneBank, M19921).5′ biotin-GCTCCATGTTTTTCCAGGTCTCGGGATGCTGCTCCCACCCCATATATATATATATATATAT-3′S-5′ biotin-HIVnef(AT)108871–89105′ biotin-GGGGTGGGAGCAGCATCCCGAGACCTGGAAAAACATGGAGCATATATATATATATATATAT-3′AS-5′ biotin-HIVgag1(AT)101484–14475′ biotin-CTTGGTTCCTCATCTGGCCTGGTGCAATAGGCCCTGCATATATATATATATATATAT-3′S-5′ biotin-HIVgag1(AT)101447–14845′ biotin-GCAGGGCCTATTGCACCAGGCCAGATGAGAGAACCAAGATATATATATATATATATAT-3′AS-5′ biotin-HIVgag2(AT)101654–16175′ biotin-GTCCTTGTCTTATGTCCAGAATGCTGGTAGGGCTATACATATATATATATATATATAT-3′S-5′ biotin-HIVgag2(AT)101617–16545′ biotin-GTATAGCCCTACCAGCATTCTGGACATAAGACAAGGACATATATATATATATATATAT-3′AS-5′ biotin-HIVgag3(AT)101864–18275′ biotin-TATGGCCGGGTCCTCCCACTCCCTGACATGCTGTCATCATATATATATATATATATAT-3′S-5′ biotin-HIVgag3(AT)101827–18645′ biotin-GATGACAGCATGTCAGGGAGTGGGAGGACCCGGCCATAATATATATATATATATATAT-3′AS is anti-sense orientation and S is sense orientation.* Nucleotide positions refer to the SIVmac239 strain20Kestler H Kodama T Ringler D Marthas M Pedersen N Lackner A Regier D Sehgal P Daniel M King N Induction of AIDS in rhesus monkeys by molecularly cloned simian immunodeficiency virus.Science. 1990; 248: 1109-1112Crossref PubMed Scopus (472) Google Scholar (GeneBank, M33262).† Nucleotide positions refer to pNL4-321Adachi A Gendelman HE Koenig S Folks T Willy R Rabson A Martin MA Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone.J Virol. 1986; 59: 284-291Crossref PubMed Google Scholar (GeneBank, M19921). Open table in a new tab AS is anti-sense orientation and S is sense orientation. For preparation of SIVgag RNA probes and SIVnef RNA probes, the gag gene fragment (GenBank no. M33262, 1309 to 2841; 1533 bp) and the nef gene fragment (GenBank no. M33262, 9333 to 10,124; 792 bp) from plasmid pSIVmac23920Kestler H Kodama T Ringler D Marthas M Pedersen N Lackner A Regier D Sehgal P Daniel M King N Induction of AIDS in rhesus monkeys by molecularly cloned simian immunodeficiency virus.Science. 1990; 248: 1109-1112Crossref PubMed Scopus (472) Google Scholar were cloned into the transcription vector pGEM-T (Promega, Madison, WI). For HIV-1 RNA probes, the gag gene fragment (GenBank no. M19921, 790 to 2292; 1503 bp) from plasmid pNL43-221Adachi A Gendelman HE Koenig S Folks T Willy R Rabson A Martin MA Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone.J Virol. 1986; 59: 284-291Crossref PubMed Google Scholar was cloned into pBluescript KS (−) (Stratagene, La Jolla, CA) and the nef gene fragment (GenBank no. M19921, 8787 to 9407; 620 bp) from pNL43-2 was cloned into pGEM-T (Promega). Sense or anti-sense biotin-labeled RNA probes were generated using T3 or T7 or SP6 RNA polymerase by an RNA labeling kit (Roche, Indianapolis, IN) according to the manufacturer's instructions as described before.9Nakajima N Sata T Hanaki K Kurata K Yoshikura H Application of hybridization AT-tailing method for detection of human immunodeficiency virus RNA in cells and simian immunodeficiency virus RNA in formalin-fixed and paraffin-embedded tissues.J Virol Methods. 1999; 81: 169-177Crossref PubMed Scopus (5) Google Scholar Each of them was confirmed to detect the SIV or HIV-1 DNA with equal efficiency in filter hybridization (data not shown). The sequence homology between SIVmac23920Kestler H Kodama T Ringler D Marthas M Pedersen N Lackner A Regier D Sehgal P Daniel M King N Induction of AIDS in rhesus monkeys by molecularly cloned simian immunodeficiency virus.Science. 1990; 248: 1109-1112Crossref PubMed Scopus (472) Google Scholar and SHIV89.6PD18Matano T Kano M Nakamura H Takeda A Nagai Y Rapid appearance of secondary immune response and protection from acute CD4 depletion after highly pathogenic immunodeficiency virus challenge in macaques vaccinated with a DNA prime/Sendai virus vector boost regimen.J Virol. 2001; 75: 11891-11896Crossref PubMed Scopus (88) Google Scholar was 100% in the gag region and 99.7% in the nef region. That between SIVmac23920Kestler H Kodama T Ringler D Marthas M Pedersen N Lackner A Regier D Sehgal P Daniel M King N Induction of AIDS in rhesus monkeys by molecularly cloned simian immunodeficiency virus.Science. 1990; 248: 1109-1112Crossref PubMed Scopus (472) Google Scholar and SIVmac32H19Rud EW Cranage M Yon J Quirk J Ogilvie L Cook N Webster S Dennis M Clarke BE Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants.J Gen Virol. 1994; 75: 529-543Crossref PubMed Scopus (130) Google Scholar was 98.7% in the gag region and 97.1% in the nef region. Formalin-fixed and paraffin-embedded tissue blocks were cut in 4-μm-thick sections, and the sections were mounted on silane-coated clean glass slides. After deparaffinization in xylene and graded ethanol, the slides were immersed in diethylpyrocarbonate-treated double-distilled water. The sections were treated with a target retrieval solution (DAKO, Carpinteria, CA) at 95°C for 40 minutes, cooled to room temperature, and washed with diethylpyrocarbonate-treated double-distilled water. Then, they were treated with 0.1 to 1 μg/ml of proteinase K (DAKO) for 15 minutes at 37°C. The proteinase K was washed out and inactivated by immersing the sections twice in 0.2% glycine-0.1 mol/L Tris-HCl (pH 7.6) for 3 minutes and twice in diethylpyrocarbonate-treated double-distilled water for 3 minutes. Finally, the sections were immersed in 100% ethanol for 2 minutes and air-dried. Deparaffinized sections were prehybridized in hybridization buffer at 37°C for 30 minutes and hybridized with anti-sense or sense RNA probes (a mixture of gag probe and nef probe) overnight at 50°C in 40 μl of hybridization buffer consisting of 50% formamide (WAKO, Tokyo, Japan); 3× standard saline citrate (SSC); 1× Denhardt's solution (WAKO); 50 mmol/L Hepes, pH 7.0; 1 mmol/L ethylenediaminetetraacetic acid; and 500 μg/ml tRNA (Roche). After hybridization the sections were washed once in 0.1× SSC (1× SSC; 150 mmol/L NaCl, 15 mmol/L sodium citrate), followed by stringent washing in 0.01× SSC for 15 minutes at 55°C twice. Then the sections were treated with 0.3% H2O2/methanol for 30 minutes to block endogenous peroxidase activities and they were immersed in 25% Blockace (Snow Brand, Tokyo, Japan)/Tris-buffered saline [TBS; 100 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl] for 60 minutes at room temperature or overnight at 4°C. For signal detection by the CSA method, the GenPoint system (DAKO) was used as follows. A 1000-fold diluted primary streptavidin-horseradish peroxidase (SA-HRP) solution was applied on each tissue section for 15 minutes at room temperature. The section was then washed three times with TBST [0.1 mol/L Tris-HCl (pH 7.5), 0.15 mol/L NaCl, 0.05% Tween 20] for 5 minutes. A solution of biotinyl tyramide was applied on the section, which was incubated for 15 minutes and then washed three times with TBST for 5 minutes. The secondary SA-HRP was applied on the section that was incubated for 15 minutes at room temperature and washed three times with TBST for 5 minutes. Signals were developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB) (Dohjin, Kumamoto, Japan) chromogen solution, followed by hematoxylin counterstaining, and observation under a light microscope. ISH-AT procedures were performed as described previously.9Nakajima N Sata T Hanaki K Kurata K Yoshikura H Application of hybridization AT-tailing method for detection of human immunodeficiency virus RNA in cells and simian immunodeficiency virus RNA in formalin-fixed and paraffin-embedded tissues.J Virol Methods. 1999; 81: 169-177Crossref PubMed Scopus (5) Google Scholar Tissue sections were covered with 300 μl of hybridization buffer consisting of 10% formamide (WAKO), 2× SSC, 1× Denhardt's solution (WAKO), 50 mmol/L NaH2PO4/Na2HPO4, 1 mmol/L ethylenediaminetetraacetic acid, and 500 μg/ml tRNA (Roche). Briefly, 2 vol of the oligonucleotide HybrAT probe (0.1 to 0.4 pmol/μl) and 1 vol of salmon sperm DNA (10 mg/ml, Sigma, St. Louis, MO) were mixed with 17 volumes of hybridization buffer and the mixture was heated at 95°C for 5 minutes. The solution in a volume of 40 μl was applied to the sections and covered with a coverglass. After hybridization at 50°C overnight in a moist chamber, the tissue was washed once in 0.1× SSC, followed by stringent washing in 0.01× SSC for 15 minutes at 55°C twice and rinsing in TBS. An appropriate size of Hybaid EasiSeal (Hybaid, Middlesex, UK) was attached to each slide so as to cover the section entirely, and the sections were saturated with ΔTth reaction buffer (10 mmol/L Tris-HCl, pH 8.9, 1.5 mmol/L MgCl2, 80 mmol/L KCl, 0.1% sodium deoxycholate, 0.1% Triton X-100, 0.5 mg/ml bovine serum albumin; Toyobo) at 60°C for 10 minutes. Then a HybrAT reaction mixture consisting of 200 μmol/L each of dATP and dTTP, 10 μmol/L of Bioin-16-dUTP (Roche), and 50 U of ΔTth DNA polymerase (Toyobo) in ΔTth reaction buffer was applied to the sections. The slides were then placed on a hot plate, OmniGene FlatBlock (Hybaid), and heated to 60°C for 30 minutes. The sections were washed with TBS and treated with 0.3% H2O2/methanol for 30 minutes. Then they were immersed in 25% Blockace/TBS for 60 minutes at room temperature or overnight at 4°C. The procedure of signal detection by the CSA method was the same as described in the RISH-CSA method. For fluorescence detection, in place of the secondary SA-HRP, streptavidin-conjugated Alexa Fluor 488 (Molecular Probes, Eugene, OR) was applied on the tissue section for 30 minutes at 37°C. Alexa Fluor 488 dye was excited with a 488-nm line of the argon-krypton laser to exhibit green fluorescence. The sections were then washed with PBS, and covered with cover glasses after application of mounting medium for fluorescence with anti-fading reagents (Vectashild; Vector Laboratories, Burlingame, CA). Imaging was performed using a confocal microscope equipped with an argon-krypton laser (LSM-MicroSystem; Zeiss, Germany). Negative controls included: 1) hybridization of lymph nodes and brain tissues from uninfected monkeys or humans, 2) hybridization with sense and irrelevant probes, 3) mock (no probe) hybridization, and 4) omission of ΔTth DNA polymerase. When there is too much target RNA or DNA in the section, omission of ΔTth DNA polymerase is unsuitable for negative control, because some signals might be obtained by only in situ hybridization using 5′ end-labeled HybrAT probe without a following elongation reaction by ΔTth DNA polymerase. After deparaffinization with xylene and ethanol, the tissue sections were rehydrated in distilled water and treated with 1 mmol/L ethylenediaminetetraacetic acid (pH 8.0) at 121°C for 10 minutes. After washing with PBS, they were immersed in 0.1 mol/L glycine-HCl buffer (pH 2.2) at room temperature for 90 minutes, washed again with PBS, and treated with 0.3% H2O2/methanol at room temperature for 30 minutes. After washing in PBS, the tissue sections were incubated in 5% normal goat serum at room temperature for 20 minutes. Excess solutions were discarded and anti-SIVgag p27 polyclonal antibody (1:3000 diluted in PBS) or anti-HIV-1gag p24 mouse monoclonal antibody (NU24, 1:4000 diluted in PBS) were applied on the sections at 4°C overnight. After washing three times with PBS, biotin-conjugated goat anti-mouse IgG (LSAB kit, DAKO) and subsequently SA-HRP (LSAB kit, DAKO) were applied on the sections. The reaction was visualized using DAB or Vector VIP (Vector Laboratories, Burlingame, CA) chromogen solution. Immunohistochemistry was applied in combination with ISH-AT-CSA to identify cells expressing viral RNA. Endogenous biotin and biotinyl tyramide after ISH-AT-CSA staining with DAB as a chromogen were blocked with avidin solution and biotin solution (Biotin Blocking System, DAKO) according to the manufacturer's instructions. Then, the tissue sections were incubated with 5% normal goat serum, followed by incubation with each mouse monoclonal anti-human cell marker antibody at 4°C overnight. The mouse monoclonal antibodies used were CD45RO (UCHL-1, DAKO), CD3 (PC3/188A, DAKO), and CD8 (NCL-CD8-295; Novocastra, Newcastle, UK) for T cells, CD68 (HAM56 for monkey, PGM1 for human; DAKO) for microglial/macrophage cells, CD20 (L26, DAKO) for B cells and CD21 (IF8, DAKO) for follicular dendritic cells (FDCs). After washing three times with PBS, biotin-conjugated goat anti-mouse IgG (DAKO) and subsequently SA-HRP (DAKO) were applied on the sections. The reaction was visualized using Vector VIP as a chromogen, followed by 2% methyl green counterstaining. For fluorescence detection, streptavidin-conjugated Alexa Fluor 568 (Molecular Probes) was used in place of SA-HRP. Alexa Fluor 568 dye was excited with a 543-nm line of the argon-krypton laser and exhibited red fluorescence. The ISH-AT-CSA signals exhibited green fluorescence. The emission patterns of the two fluorescent labels were collected separately and the data were overlaid on a computer to generate two-color images. First, we attempted to detect viral RNA using the formalin-fixed and paraffin-embedded autopsy tissues from rhesus monkeys experimentally infected with SHIV89.6P
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