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

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.