Immunocytochemical Analysis of Cyclic Nucleotides

J. deVente1, H.W.M. Steinbusch1

1 Maastricht University, Maastricht, The Netherlands
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 10.7
DOI:  10.1002/0471140856.tx1007s02
Online Posting Date:  May, 2001
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Abstract

Visualization of second messenger molecules is a powerful approach to study the role of second messengers in relation to the (sub)cellular localization and/or kinetics of the second messenger response. This unit describes the appropriate fixation procedures for the cyclic nucleotides cAMP and cGMP and the use of specific antibodies to localize these molecules.

     
 
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Table of Contents

  • Basic Protocol 1: Immunocytochemistry for Cyclic Nucleotides Using In Vitro Fixed Brain Slices
  • Alternate Protocol 1: In Vivo Fixation for Cyclic Nucleotide Immunocytochemistry
  • Support Protocol 1: Preparation and Characterization of Antisera to Conjugated Cyclic Nucleotides
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Immunocytochemistry for Cyclic Nucleotides Using In Vitro Fixed Brain Slices

  Materials
  • Animal source of tissue
  • Krebs buffer, oxygenated with 5% CO 2/95% O 2 (see recipe), ice cold
  • Cyanoacrylate glue
  • Drug or toxin to be tested for effects on cGMP or cAMP levels
  • Formaldehyde fixative (see recipe), ice cold
  • Formaldehyde fixative containing 10% sucrose (see recipe)
  • 10% (w/v) sucrose
  • Optimal cutting (OCT) compound (cryostat embedding medium)
  • Acrolein fixative (see recipe)
  • Glycine
  • 0.1 M phosphate buffer, pH 7.0, containing 10% sucrose
  • TBS (see recipe)
  • 10 mM NaCNBH 3 in 0.1 M sodium acetate, pH 7.45
  • Anti‐cGMP‐formaldehyde‐protein or anti‐cAMP‐acrolein‐protein antiserum (see protocol 3)
  • TBS‐T: recipeTBS containing 0.3% Triton X‐100
  • Secondary and tertiary antibodies conjugated with reporter molecules (e.g., FITC or Cy3; see Troubleshooting)
  • 1:3 (v/v) recipeTBS/glycerol
  • DAB staining solution (see recipe)
  • Grid glued to a firmly fitting teflon support ring into the multiwell tissue culture plate
  • 12‐well plates
  • Vibrating tissue knife: Vibratome (Technical Products) or Vibroslicer (WPI)
  • Cryostat
  • Chrome‐alum‐coated glass microscopic slides

Alternate Protocol 1: In Vivo Fixation for Cyclic Nucleotide Immunocytochemistry

  • Animal source of tissue
  • Pentobarbital sodium (Nembutal)
  • Krebs buffer (see recipe) prepared without calcium, oxygenated with 5% CO 2/95% O 2
  • Drug to be tested for effects on cGMP or cAMP levels
  • Formaldehyde fixative (see recipe)
  • TBS (see recipe) containing 10% (w/v) sucrose
  • 0.9% (w/v) NaCl ( appendix 2A)
  • Chrom‐alum‐ or lysine‐coated slides

Support Protocol 1: Preparation and Characterization of Antisera to Conjugated Cyclic Nucleotides

  Materials
  • Carrier protein (e.g., bovine thyroglobulin, BSA, hemocyanin)
  • 3′,5′ cGMP or 3′,5′cAMP (sodium salt)
  • 0.2 M sodium phosphate buffer, pH 7.0 ( appendix 2A)
  • Formaldehyde fixative (see recipe)
  • 0.1 M sodium phosphate buffer, pH 7.0 ( appendix 2A), 4°C
  • 0.1 M sodium acetate, pH 4.75 ( appendix 2A)
  • Acrolein fixative (see recipe)
  • Glycine
  • NaCNBH 3
  • Rabbits (or other species in which to raise antibodies)
  • 0.9% (w/v) NaCl ( appendix 2A)
  • Complete and incomplete Freund's adjuvant
  • Fixative‐treated carrier protein
  • Glycerol
  • Blocking solution: 1% (w/v) gelatin and 1% (w/v) nonfat dry milk in water
  • TBS (see recipe)
  • TBS‐T: recipeTBS containing 0.3% (v/v) Triton X‐100
  • Anti–rabbit Ig (secondary antibody)
  • Rabbit peroxidase‐antiperoxidase antibody
  • 0.05 M Tris⋅Cl, pH 7.0 ( appendix 2A)
  • DAB staining solution (see recipe)
  • Dialysis equipment
  • Homogenizer, either standard Potter system or high‐speed‐blade (Polytron‐type) system
  • Nitrocellulose or nylon membrane
  • Glass capillary micropipets
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Figures

Videos

Literature Cited

Literature Cited
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Key References
   de Vente et al., 1987. See above.
   Describes the preparation of cGMP‐formaldehyde conjugates and gives details about stability, retention, and characterization.
   de Vente et al., 1989. See above.
   Describes at length the various methods for studying the specificity of these antibodies.
   de Vente et al., 1993. See above.
   Describes for the first time preparation of cAMP‐acrolein conjugates and first characterization of these antibodies.
   Ewer et al., 1994. See above.
   Describes the first application of the antibody in insect tissue.
   Shuttleworth et al., 1993. See above.
   Provides an example of the importance of the choice of phosphodiesterase inhibitor.
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