A New Approach to Immunocytochemistry of 3',5'-cyclic Guanosine Monophosphate: Preparation, Specificity, and Initial Application of a New Antiserum Against Formaldehyde-Fixed 3',5'-cyclic Guanosine Monophosphate

Neuroscience. 1987 Jul;22(1):361-73. doi: 10.1016/0306-4522(87)90226-0.

Abstract

The development of a new 3',5'-cyclic guanosine monophosphate (cGMP) antiserum was initiated starting from the following considerations: (a) adequate fixation of cGMP is a prerequisite for a reliable demonstration of soluble cGMP, and (b) fixation might influence the specificity of the immunocytochemical demonstration of cGMP. Therefore, cGMP-protein conjugate was prepared in a way which equals tissue fixation. cGMP was coupled to bovine thyroglobulin using formaldehyde. Antibodies against this conjugate were raised in rabbits. The specificity of the antisera was evaluated in a gelatin model system. No immunoreactivity was observed with nucleotides other than cGMP or with rabbit preimmune sera. Immunoinhibition experiments showed that only the cGMP-formaldehyde-thyroglobulin conjugate and, to a lesser extent free cGMP, absorbed onto the antiserum. In rat brain an extensive localization of cGMP-immunostaining was found. Examples are hippocampus CAI and CAII, and cortical layers II and V. No cGMP-immunostaining was found in the cerebellum. In vitro incubated superior cervical ganglia showed cGMP-immunostaining in the large postganglionic neuronal cell bodies; this cGMP-immunostaining increased upon incubation of the ganglia in iso-osmolar 100 mM K+. In conclusion, we prepared a new-type highly specific antiserum against cGMP, suitable to demonstrate cGMP-immunoreactivity in tissue material.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / anatomy & histology*
  • Brain Mapping
  • Cyclic AMP / metabolism
  • Cyclic GMP / metabolism*
  • Fluorescent Antibody Technique*
  • Ganglia, Sympathetic / anatomy & histology*
  • Radioimmunoassay
  • Rats

Substances

  • Cyclic AMP
  • Cyclic GMP