Cyclic Nucleotide Immunohistochemistry

AbstractIt has been reported that cyclic nucleotide immunohistochemistry fails to localize all pools of cyclic nucleotides and to detect the changes in cyclic nucleotide concentration when applied to unfixed brain tissue sections. Losses of cyclic nucleotides during antibody incubations have been demonstrated, but it has not been determined whether such losses are due entirely to extraction or if phosphodiesterase activity contributes to the loss. We also found that cyclic nucleotides are extracted during the incubations required by the procedure but report that the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) reduces cyclic nucleotide losses.The application of the unlabelled antibody-enzyme technique and the inclusion of IBMX in incubation solutions permitted the detection of cyclic GMP in all layers of rat cerebellar cortex. The administration of ethanol (4g/kg, per os) ubiquitously reduced cyclic GMP staining.