In Vitro Autoradiography

Michael J. Kuhar1

1 Yerkes Regional Primate Research Center, Atlanta, Georgia
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 8.1
DOI:  10.1002/0471141755.ph0801s00
Online Posting Date:  May, 2001
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The light‐microscopic localization of receptors and other molecules by autoradiography of radioligand binding in tissue sections provides a high degree of anatomical resolution and the ability to associate even small quantities of binding sites with anatomical structures. The basic procedure in this unit involves incubating slide‐mounted tissue sections with radioligands, washing and drying of the sections with specifically bound ligands under conditions that preserve ligand binding, and visualizing and quantifying the binding sites in the tissues. While the procedures were developed for drug and neurotransmitter receptors, any binding site can be visualized with a sufficiently selective, high‐affinity radioligand. If optimal conditions for radioligand binding to sites in slide‐mounted tissue sections are already known, the basic protocol in this unit can be used to create autoradiograms. However, if a radioligand is being studied for the first time, guidelines are provided on optimizing experimental parameters for binding, washing, and drying. The light‐microscopic localization of receptors and other molecules by autoradiography of radioligand binding in tissue sectio.

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Table of Contents

  • Basic Protocol 1: Receptor/Binding‐Site Autoradiography
  • Support Protocol 1: Determining Optimal Autoradiography Conditions for a New Radioligand
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Receptor/Binding‐Site Autoradiography

  • Target tissue (e.g., brain)
  • Mounting medium (e.g., Tissue‐Tek from Fisher or homemade brain paste)
  • Incubation medium: appropriate buffer with radioligand at appropriate concentration (as determined by homogenate binding experiments; see Chapter )
  • Wash medium: appropriate buffer (as determined in protocol 2 or homogenate binding experiments) without radioligand
  • Cryostat chucks: metal platforms for supporting specimens during sectioning
  • Microtome/cryostat operating between −10° and −25°C (with microtome knives and appropriate accessories)
  • Gelatin‐subbed glass microscope slides (see recipe)
  • Source of cool, dry air (e.g., setup illustrated in Fig. , panel B)
  • Hair dryer set to room temperature
  • Slide boxes containing desiccant
  • Suitable radioisotope standards (e.g., Amersham)
  • Autoradiography cassette and cardboard sheet cut to fit
  • Double‐sided tape
  • Autoradiography film (appropriate for radiolabel used, e.g., tritium‐sensitive sheet film; Kodak or Amersham)
  • Image‐analysis equipment (microscope‐ or video‐camera based)

Support Protocol 1: Determining Optimal Autoradiography Conditions for a New Radioligand

  • Small circles of filter paper (e.g., Whatman B or C)
  • Scintillation cocktail and vials
  • Scintillation counter
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Literature Cited

Literature Cited
  Boast, C., Snowhill, E.W., Altar, C.A. (eds.) 1986. Quantitative Receptor Autoradiography. Alan R. Liss, New York.
   Cuatrecasas, C. 1971. Insulin‐receptor interactions in adipose tissue cells: Direct measurement and properties. Proc. Natl. Acad. Sci. U.S.A. 68:1264‐1268.
   Ehn, E. and Larsson, B. 1979. Properties of an antiscratch‐layer‐free x‐ray film for the autoradiographic registration of tritium. Sci.Tools. 26:24‐29.
   Gerlach, J.L. and McEwen, B.S. 1972. Rat brain binds adrenal steroid hormone: Radioautography of hippocampus with corticosterone. Science. 175:1133‐1136.
   Herkenham, M. and Pert, C.B. 1982. Light microscopic localization of brain opiate receptors: A general autoradiographic method which preserves tissue quality. J. Neurosci. 2:1129‐1149.
   Herkenham, M. and Sokoloff, L. 1984. Quantitative receptor autoradiography: Tissue defatting eliminates differential self‐absorption of tritium radiation in gray and white matter of brain. Brain Res. 321:363‐368.
   Kuhar, M.J. and DeSouza, E.B. 1988. Autoradiographic imaging: Localization of binding sites other than neurotransmitter receptors. In Visualization of Brain Functions. (D. Ottoson and W. Rostene, eds.) pp. 57‐66. Stockton Press, New York.
   Kuhar, M.J. and Unnerstall, J.R. 1982. In vitro receptor autoradiography: Loss of label during ethanol dehydration and preparative procedures. Brain Res. 244:178‐181.
   Kuhar, M.J. and Unnerstall, J.R. 1985. Quantitative receptor mapping by autoradiography: Some current technical problems. Trends Neurosci. 8:49‐53.
   Kuhar, M.J. and Unnerstall, J.R. 1990. Receptor autoradiography. In Methods in Neurotransmitter Receptor Analysis (H.I. Yamamura, S.J. Enna, and M.J. Kuhar, eds.) pp. 177‐218. Raven Press, New York.
   Penney, J.B., Pan, H.S., Young, A.B., Frey, K.A., and Dauth, G.W. 1981. Quantitative autoradiography of 3H‐muscimol binding in rat brain. Science. 214:1036‐1038.
   Rainbow, T.C., Bleisch, W.V., Bigeon, A., and McEwen, B.S. 1982. Quantitative densitometry of neurotransmitter receptors. J. Neurosci. Methods. 5:127‐138.
   Roth, L.J. and Stumpf, W.E. 1969. Autoradiography of Diffusible Substances. Academic Press, New York.
   Roth, L.J., Diab, I.M., Watanabe, M., and Dinerstein, R.J. 1974. A correlative radioautographic, fluorescent, and histochemical technique for cytopharmacology. Mol. Pharmacol. 10:986‐998.
   Rotter, A., Birdsall, N.J.M., Burgen, A.S.C., Field, P.M., Hulme, E.C., and Raisman, G. 1979. Muscarinic receptors in the central nervous system of the rat. I. Technique for autoradiographic localization of the binding of [3H] propylbenzilylcholine mustard and its distribution in the forebrain. Brain Res. Rev. 1:141‐166.
   Snyder, S.H. and Bennett, J.P. Jr. 1976. Neurotransmitter receptors in the brain: Biochemical identification. Annu. Rev. Physiol. 38:153‐175.
   Whitehouse, P.J., Lynch, D., and Kuhar, M.J. 1984. Effects of postmortem delay and temperature on neurotransmitter receptor binding in a rat model of the human autopsy process. J. Neurochem. 43:553‐559.
   Young, W.S. and Kuhar, M.J. 1978. Opiate receptor autoradiography: In vitro labeling of tissue slices. In Characteristics and Function of Opioids (V. Ree and L.L. Terenius, eds.) pp. 451‐452. Elsevier/North‐Holland, Amsterdam.
   Young, W.S., III and Kuhar, M.J. 1979. A new method for receptor autoradiography: 3H‐opioid receptor labeling in mounted tissue sections. Brain Res. 179:255‐270.
Key Reference
   Kuhar and Unnerstall, 1990. See above.
  An excellent, detailed, and thorough review of the topic.
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