Measurement of In Situ Hybridization

Shelley B. Hoover1, Cecil H. Fox1, Jim Bahre2, Michele Cottler‐Fox3, Cynthia Sung4

1 Molecular Histology Laboratory, Inc., Gaithersburg, Maryland, 2 Fuji Medical Systems, USA, Stamford, Connecticut, 3 University of Maryland Greenebaum Cancer Center, Baltimore, Maryland, 4 Bioengineering and Physical Science Program/National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.12
DOI:  10.1002/0471142727.mb1412s49
Online Posting Date:  May, 2001
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Abstract

Hybridization of labeled specific molecular probes to nucleic acids in tissues allows geometric and functional location of gene expression or of foreign genome sequences. Estimates of amounts and location of target nucleic acid sequence can be made with phosphor storage imaging and molecular controls.

     
 
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Table of Contents

  • Basic Protocol 1: Determining the Distribution of Radiolabeled Heteroduplexes in In Situ Hybridizations by Phosphor Storage Imaging
  • Support Protocol 1: Producing a Reference System
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determining the Distribution of Radiolabeled Heteroduplexes in In Situ Hybridizations by Phosphor Storage Imaging

  Materials
  • Reference standards (clots; see protocol 2) hybridized with the same radiolabeled probe used for in situ hybridization
  • Radiation standards consisting of 14C embedded in plastic (suitable for 35S or 33P; may be purchased on a microscope slide from American Radiolabeled Chemicals; http://www.arc‐inc.com)
  • Hybridized slides from in situ hybridization (Chapter 14)
  • Phosphor storage imager (e.g., Fuji BAS 5000) with phosphor plates
  • Light box or high‐intensity light for erasing phosphor plates
  • Manila folders
  • Double‐sided adhesive tape (Scotch brand)
  • Plastic wrap (e.g., Saran wrap)
  • Image processing software: NIH Image (available for free from the NIH website; http://rsb.info.nih.gov/nih‐image/Default.html) or MCID (Imaging Research)

Support Protocol 1: Producing a Reference System

  Materials
  • recipeRadiolabeled standards: sample of RNA, DNA, or virion suspension (see recipe)
  • recipeFibrin glue (see recipe)
  • recipeThrombin, USP (see recipe; dilute to 10,000 U/ml)
  • recipe1.3 M formaldehyde solution without buffers or salt (see recipe)
  • 70% ethanol
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Figures

Videos

Literature Cited

Literature Cited
   Cottler‐Fox, M. and Fox, C.H. 1991. Examining cells for infectious agents: a novel approach. J. Infect. Dis. 164:1239‐1240.
   Fox, C.H. and Cottler‐Fox, M. 1993. In situ hybridization for detection of HIV RNA. In Current Protocols in Immunology (J. Coligan, A., Kruisbeek, D., Margulies, E., Shevach, W., Strober, eds.) pp. 12.8:1‐12.8.21. John Wiley & Sons, New York.
   Fox, C.H., Hoover, S., Currall, V.R., Bahre, H.J. and Cottler‐Fox, M. 1994. HIV in infected lymph nodes. Nature 370:256.
   Gall, J.G. and Pardue, M.L. 1971. Nucleic acid hybridization in cytological preparations. Methods Enzymol. 38:470‐480.
   Miyahara, J. 1989. Visualizing things never seen before: The imaging plate, a new radiation sensor. Chemistry Today Oct. 1989:29‐36.
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