Autoradiographic Localization of Growth Factor Receptors in Neuronal Tissues

Sylvain Doré1, Satyabrata Kar1, Daniel Auld1, Jean‐Guy Chabot1, Remi Quirion2

1 Douglas Hospital Research Center and McGill University, Montreal, Canada, 2 Douglas Hospital Research Center, Verdun, Canada
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 8.2
DOI:  10.1002/0471141755.ph0802s03
Online Posting Date:  May, 2001
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Abstract

The physiological and pharmacological effects of growth factors, like those of neurotransmitters and neuromodulators, are mediated by binding to specific receptor sites. Using radiolabeled molecules with high specific activity, such as the endogenous ligand itself, it is possible to measure receptor binding in membrane preparations. This technique provides a means to characterize the selectivity and specificity of the receptors for a particular growth factor. Additionally, ligand‐binding assays make it possible to study in detail the structure‐activity relationships and mechanisms of action of growth factors. As an example of this methodology, this unit describes the use of both film and emulsion autoradiography to study the pharmacological characteristics and cellular distribution profile of insulin‐like growth factor receptors in the central nervous system. For the sake of clarity, in vitro film autoradiography in tissue sections and liquid emulsion autoradiography in cultured neurons are discussed separately. The physiological and pharmacological effects of growth factors, like those of neurotransmitters and neuromodulators, are medi.

     
 
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Table of Contents

  • Basic Protocol 1: In Vitro Receptor Autoradiography in Tissue Sections Using Dry Films
  • Alternate Protocol 1: In Vitro Receptor Autoradiography in Cultured Neurons Using Liquid Emulsion
  • Support Protocol 1: Preparation of Gelatin‐Coated Slides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Vitro Receptor Autoradiography in Tissue Sections Using Dry Films

  Materials
  • Surgically or pharmacologically treated animal
  • Normal control animals
  • 2‐Methylbutane, −40°C
  • Gelatin‐coated slides (see protocol 3), precooled to −15° to −18°C
  • IGF‐I, IGF‐II, or insulin incubation buffer (prepared fresh; see recipe recipe recipe)
  • IGF‐I buffer recipe prepared without radioligand (for IGF‐I assay only)
  • Recombinant human IGF‐I (rhIGF‐I; ICN Biomedicals), IGF‐II (rhIGF‐II; Sigma), or porcine insulin (Peninsula Labs)
  • 50 mM Tris⋅Cl, pH 7.4 ( appendix 2A; for IGF‐I or IGF‐II assay) or 10 mM HEPES, pH 7.4 (for insulin assay), 4°C
  • Dry ice
  • Embedding medium (e.g., Lipshaw)
  • 245 × 245–mm polypropylene Bioassay dish (Cole‐Parmer)
  • X‐ray film (Hyperfilms, Amersham; or Ultrofilm, NEN Life Science Products)
  • Radiolabeled standard or microscales for calibration and quantification (Amersham)
  • X‐ray film cassettes
  • Computerized image‐analysis system (e.g., MCID; Imaging Research)

Alternate Protocol 1: In Vitro Receptor Autoradiography in Cultured Neurons Using Liquid Emulsion

  Materials
  • Appropriate cultured neurons grown on gelatin/poly‐L‐lysine‐coated coverslips or culture chambers (see protocol 3 for coating procedure) in DMEM with B‐27 supplement (Life Technologies)
  • DMEM without B‐27 supplement (Life Technologies)
  • In vitro incubation buffer (prepared fresh; see recipe)
  • Unlabeled rhIGF‐I, rhIGF‐II, or porcine insulin
  • Phosphate‐buffered saline (PBS)
  • 2.5% glutaraldehyde (Sigma)
  • 50%, 70%, 90%, and 100% ethanol
  • Superglue or DPX mounting medium
  • NTB‐2 liquid emulsion (Eastman Kodak)
  • 60 mM ammonium acetate or distilled H 2O
  • Nissl or hematoxylin/eosin stain
  • Xylene
  • Permount
  • 42°C water bath
  • Slide cytomailers
  • Black slide‐storage box
  • Computerized image analysis system (e.g., MCID; Imaging Research) attached to a bright‐field microscope

Support Protocol 1: Preparation of Gelatin‐Coated Slides

  Materials
  • Detergent solution
  • 100% ethanol
  • Gelatin powder
  • Chrom alum (chromium potassium sulfate; Sigma)
  • Whatman paper
  • Slides with stainless steel racks and storage boxes
  • Boiling water bath
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Figures

Videos

Literature Cited

Literature Cited
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