An evaluation of immunohistochemistry for studying cyclic nucleotides in the central nervous system, with particular reference to guanosine 3',5'-monophosphate

Abstract

An immunofluorescent technique has been developed with the aim of histochemically localizing cyclic AMP and cyclic GMP in the C.N.S., and determining the cellular sites where biochemical changes in tissue levels occur.

Initial experiments revealed that the inmunoglobulin fraction of non -immune sera bound non -specifically to C.N.S. tissue, via a weak charge attachment between IgG and basic tissue proteins.

Specific antibodies were raised in rabbits by immunization with succinyl cyclic nucleotide- protein conjugates, and radioiunological techniques with[3H]tracers were used to study binding characteristics. Of the large number of cyclic GMP antibodies studied in detail, specific staining of astrocytic fibres and capillaries (a contrasting localization to that of cyclic AMP), was found with only a small number of antibodies, although these could not be identified on the basis of titre, avidity or specificity however.

Differences between RIA and immunohistochemistry, and different staining patterns with individual cyclic GMP antibodies, have been discussed as resulting from stereochemical differences between free and tissue -bound nucleotide. This may also explain why cyclic nucleotide antibodies have satisfied the criteria of specificity for RIA, but not for immunofluorescence.

'In vivo' and 'in vitro' biochemical techniques, coupled with cyclic GMP immunofluorescence, failed to localize semi -quantitative changes in intensity and /or distribution of staining, under conditions where total nucleotide levels were significantly altered. - iv - Quantifying cyclic nucleotide losses from frozen tissue sections, it was determined that more than 80% of cyclic GMP was lost during buffer -washing, and that the tissue -bound pool was unchanged when total levels were elevated - a possible explanation for the inability to localize biochemical changes using i mm nofluorescence.

Quantifying cyclic nucleotide losses from frozen tissue sections, it was determined that more than 80% of cyclic GMP was lost during buffer -washing, and that the tissue -bound pool was unchanged when total levels were elevated - a possible explanation for the inability to localize biochemical changes using immnofluorescence.

Recently developed antibodies to cyclic GMP- dependent protein kinase, and other cyclic nucleotide receptor proteins, were used to investigate the binding, and determine the function of, the pool of cyclic nucleotides localized in C.N.S. tissue sections by immunofluorescence.