In Situ Hybridization for Detection of HIV RNA

Cecil H. Fox1, Michele Cottler‐Fox2

1 Molecular Histology, Gaithersburg, Maryland, 2 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 12.8
DOI:  10.1002/0471142735.im1208s06
Online Posting Date:  May, 2001
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Abstract

In HIV studies, in situ hybridization can be used for identifying virion RNA, mRNA being produced for virion packaging, and proviral DNA in the cytoplasm or integrated in the nucleus. This unit focuses primarily on identifying virion RNA, because this is the most sensitive means by which in situ hybridization can be employed to detect HIV expression. In situ hybridization, as developed for HIV RNA detection, involves several protocols: (1) preparation of a radioactive or nonradioactive RNA probe; (2) in situ hybridization of probe to cells and paraffin sections of tissue; (3) detection of radiolabeled probe by emulsion autoradiography; (4) development, staining, and mounting of slides; and finally (5) examination of slides by bright‐field, dark‐field, specular reflectance, or laser‐scanning confocal microscopy. The protocols presented in this unit describe a setup involving up to 150 slides.

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of Radiolabeled RNA Probes
  • Basic Protocol 2: In situ Hybridization to Cells and Paraffin Sections of Tissue
  • Basic Protocol 3: Detection of Radioactive RNA Probe by Emulsion Autoradiography
  • Basic Protocol 4: Staining and Mounting of Autoradiographed Slides
  • Basic Protocol 5: Microscopic Examination of In Situ Hybridization Slides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of Radiolabeled RNA Probes

  Materials
  • recipe[35S]CTP (1500 mCi/mmol; Du Pont NEN #NEG‐064C) or other labeled nucleotide
  • RNA synthesis kit containing transcription buffer, DTT, RNase‐free water, UTP, ATP, and GTP (Promega #P1270)
  • Linearized plasmid containing HIV DNA insert (Table 12.8.1)
  • RNAsin (Promega #N2113)
  • recipeDistilled, deionized H 2O treated with diethylpyrocarbonate (DEPC‐treated water)
  • SP6 and T7 RNA polymerase (unit 10.9; Promega)
  • RNase‐free DNase (Promega RQ1 #M6101)
  • recipeAlkaline hydrolysis buffer
  • 3 M sodium acetate, pH 5.2 (adjust with glacial acetic acid)
  • NenSorb 20 chromatography kit (Du Pont NEN #NLP‐022)
  • recipeReagent A
  • 70% and 100% ethanol
  • Yeast tRNA or mRNA (Sigma)
  • recipeTris·Cl‐EDTA buffer, pH 7.4
  • 5 M NaCl
  • 20% and 5% (v/v) trichloroacetic acid (TCA)
  • 1.5‐ml microcentrifuge tubes
  • 6‐ml syringe
  • Liquid scintillation counter
  • 11 × 100–mm glass test tube (7‐ml)
  • 25‐mm‐diameter, 0.45‐µm pore size nitrocellulose filter (Schleicher & Schuell #BA85)
  • Filter funnel (e.g., Millipore Filter XX 1002514 funnel apparatus), mounted on vacuum flask
  • Adjustable‐temperature water bath
NOTE: When 35S‐labeled probes are prepared, it is very important to add 10 mM DTT to all solutions containing labeled nucleotides, particularly after any step that may inactivate DTT (e.g., precipitation, column chromatography, and boiling). In addition, extreme care should be taken to prevent RNase contamination of reagents.

Basic Protocol 2: In situ Hybridization to Cells and Paraffin Sections of Tissue

  Materials
  • 75 × 25–mm microscope slides containing tissue or cytospin slides containing cell samples
  • Xylene
  • 100% and 95% ethanol
  • recipeDistilled, deionized H 2O treated with diethylpyrocarbonate (DEPC‐treated water), 37°C and room temperature
  • 0.2 N HCl prepared in recipeDEPC‐treated H 2O
  • Proteinase K (Sigma #P4914)
  • recipe1 M Tris·Cl, pH 7.4 ( appendix 2A)
  • 1 M CaCl 2
  • Triethanolamine (Fluka #90290) buffer, pH 8.0 (adjust pH with 10 M KOH just before use)
  • Succinic anhydride (Fluka #14089)
  • Acetic anhydride (Fluka #09689)
  • recipePrehybridization solution
  • Dextran sulfate (Sigma #D6001)
  • recipeDeionized formamide
  • recipeHybridization cocktail
  • Sense and antisense probes ( protocol 1first basic protocol)
  • recipe2× SSC wash solution
  • recipeTriton wash solution
  • recipe0.1× SSC wash solution
  • recipeFormamide wash solution
  • recipeRNase digestion solution
  • recipe0.3 M ammonium acetate/70% ethanol
  • recipe0.3 M ammonium acetate/95% ethanol
  • recipe1 M DTT (prepared immediately before use)
  • Slide racks (1 rack for every 50 slides)
  • Staining jars to hold racks
  • Sterile glass test tube
  • 4.5‐ml plastic Nunc tube with cap
  • 500‐µl repeating pipettor
  • Gauze
  • Coverslips, No. 11/2, in 22‐, 30‐, 40‐, and 50‐mm lengths (22 mm widths)
  • Rubber cement (Carter's #845)
  • 20‐ml plastic syringe
  • Heavy duty aluminium foil
  • 4‐liter (1‐gallon) plastic jugs
  • Heat‐sealable plastic bags (e.g., Scotchpack)
  • Adjustable‐temperature water baths
  • Single‐edge razor blade

Basic Protocol 3: Detection of Radioactive RNA Probe by Emulsion Autoradiography

  Materials
  • Peel‐A‐Way plastic slide holders (Polysciences)
  • Slides containing hybridized probe (second protocol 2basic protocol)
  • NTB‐3 emulsion (Eastman Kodak; stored at 4°C)
  • D19 developer (Eastman Kodak; prepared the day before use and stored at 4°C)
  • Fixer (Eastman Kodak; prepared the day before use and stored at 4°C)
  • 45°C water bath
  • Slide holders and dark box for autoradiography
  • Desiccant
  • Darkroom

Basic Protocol 4: Staining and Mounting of Autoradiographed Slides

  Materials
  • Autoradiographed, hydrated in situ hybridization slides (third protocol 3basic protocol)
  • Hematoxylin stain (Harris's old formula; American HistoLabs)
  • 70%, 95%, and 100% ethanol
  • 0.1% eosin Y/0.1% phloxine prepared in 95% ethanol
  • 3% ammonium hydroxide in 70% ethanol
  • 1% (v/v) HCl in 70% ethanol
  • Hemo‐De (a less toxic terpene solvent) or xylene
  • Mounting medium: Permount (Fisher) or Canada balsam ( Fluka)
  • No. 11/2 coverslips in assorted lengths
  • Staining jars

Basic Protocol 5: Microscopic Examination of In Situ Hybridization Slides

  Materials
  • Research‐model microscope (e.g., Leica, Nikon, Olympus, Zeiss) equipped with:
  •  Phase‐contrast and/or dry dark‐field condenser
  •  Incident‐light mercury arc illuminator
  •  Dichroic polarizer
  •  Polarizing analyzer
Attachment cameraNOTE: The mercury arc illuminator, dichroic polarizer, and polarizing analyzer are required for specular reflectance microscopy only.For first inspection of slides it is best to use a low‐power objective (4× to 16×) with a makeshift dark‐field illuminator made by turning a phase condenser to the No. 3 annulus (contact your microscope manufacturer's technical representative for help). Dark‐field microscopy is one of the better techniques for minimizing in situ hybridization artifacts when analyzing HIV specimens.For tissues with large areas of intense signals (e.g., lymph nodes), use a 1× objective with a special condenser called a Hinsch box. For a detailed explanation on photographing such specimens, see Fox and Dreyfuss ( ).Once HIV‐positive cells have been located by dark‐field microscopy, they can be further inspected by incident‐light polarization using spectral reflectance microscopy. Spectral reflectance is a microscopic technique that is particularly useful for excluding artifacts of HIV in situ hybridization specimens; it is relatively simple but must be explained and demonstrated by your technical representative. Using incident‐light polarization causes the silver grain pattern—which is characteristic of HIV infection—to appear as green dots against a light green background (the green comes from the 546‐nm emission line of the mercury arc). The advantage of this technique is that suspect cells can be either transilluminated to show morphology, and/or epiilluminated to show silver grains, without having to change condensers.The best objectives for this type of microscopy are oil immersion 16×, 25×, 40×, or 63×. If microscope objectives are purchased with iris diaphragms, high‐quality objectives can be used for bright‐field, dark‐field, or spectral reflectance.Confocal microscopes offer a few advantages over conventional microscopes, but at enormous expense. Use of these should be considered in special cases only.The following guidelines should be followed when examining in situ hybridization slides.
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Figures

Videos

Literature Cited

Literature Cited
   Awgulewitsch, A., Utset, M.F., Hart, C.P., McGinnis, W., and Ruddle, F.H. 1986. Spatial restriction in expression of a mouse homeobox locus within the central nervous system. Nature. 320:328‐335.
   Brahic, M. and Haase, A.T. 1978. Detection of viral sequences of low reiteration frequency by in situ hybridization. Proc. Natl. Acad. Sci. U.S.A. 75:6125‐6129.
   Cottler‐Fox, M. and Fox, C.H. 1991. Examining cells for infectious agents: A novel approach. J. Infect. Dis. 164:1239‐1240.
   Cox, K.H., DeLeon, D.V., Angerer, R.C. 1984. Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev. Biol. 101:485‐502.
   Fox, C.H. and Dreyfuss, R. 1992. Photography and molecular biology: In situ hybridization autoradiography and immunogold. J. Biol. Photogr. 60:39‐44.
   Fox, C.H., Johnson, F., Whiting, J., and Roller, P.B. 1985. Formaldehyde fixation. J. Histochem. Cytochem. 33:845‐853.
   Gall, J.G. and Pardue, M.L. 1968. Nucleic acid hybridization in cytological preparations. Methods Enzymol. 38:470‐480.
   Singer, R.H. and Ward, D.C. 1982. Actin gene expression visualized in a chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog. Proc. Natl. Acad. Sci. U.S.A. 79:7331‐7335.
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