Histochemistry

, Volume 91, Issue 5, pp 401–412 | Cite as

Formaldehyde fixation of cGMP in distinct cellular pools and their recognition by different cGMP-antisera

An immunocytochemical study into the problem of serum specificity
  • J. de Vente
  • J. Schipper
  • H. W. M. Steinbusch
Article

Summary

Three different antisera raised against the same formaldehyde fixed cGMP conjugate were tested for their specificity in two non-biological and two biological model systems. The first non-biological model system was based on nucleotides fixed to gelatin by formaldehyde and the other non-biological model was nitrocellulose paper as a carrier for nucleotides coupled to proteins by formaldehyde. All antisera proved specific for cGMP in both models. As biological models we used the in vitro incubated hippocampus slice and the in vitro incubated aortic ring. In hippocampus slices all three antisera showed cGMP-producing cells after atrial natriuretic factor stimulation. However, there were significant differences in the visualization of cGMP-immunoreactivity between the three antisera when sodium nitroprusside or potassium were used to stimulate cGMP production. Nevertheless, these differential staining patterns all showed cGMP-immunoreactivity using the conventional immunocytochemical control tests. In the aorta ring all three antisera showed the same strong increase in cGMP-immunoreactivity after in vitro stimulation with sodium nitroprusside. These results were corroborated by biochemical assay of cGMP. We conclude that these three antisera all demonstrate cGMP-immunoreactivity in the biological models used. The different staining patterns that occur are caused by differences in the microchemical milieu of the formaldehyde-fixed cGMP. The use of different antibodies to cGMP may give information about this microchemical milieu which may eventually contribute to a better anderstanding of different intracellular cGMP pools.

Keywords

Formaldehyde Gelatin Staining Pattern Nitroprusside Sodium Nitroprusside 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Ariano MA, Matus AI (1981) Ultrastructural localization of cyclic GMP and cyclic AMP in rat striatum. J Cell Biol 91:287–292Google Scholar
  2. Ariano MA, Butcher LL, Appleman MM (1980) Cyclic nucleotides in the rat caudate-putamen complex: histochemical characterization and effects of deafferentiation and kainic acid infusion. Neuroscience 5:1269–1276Google Scholar
  3. Atassi MZ (1984) Antigenic structures of proteins. Their determination has revealed important aspects of immune recognition and generated strategies for synthetic mimicking of protein binding sites. Eur J Biochem 145:1–20Google Scholar
  4. Berkenbosch F, Tilders FJH (1987) A quantitative approach to cross-reaction problems in immunocytochemistry. Neuroscience 23:823–826Google Scholar
  5. Berkenbosch F, Linton EA, Tilders FJH (1986a) Colocalization of peptide histidine isoleucine amine and corticotropin-releasing factor immunoreactivity in neurons of the rat hypothalamus: a surprising artefact. Neuroendocrinology 44:338–346Google Scholar
  6. Barkenbosch F, Schipper J, Tilders FJH (1986b) Corticotropin-releasing factor immunostaining in the rat spinal cord and medulla oblongata: an unexpected form of cross-reactivity with substance P. Brain Res 399:87–96Google Scholar
  7. Braas KM, Newby AC, Wilson VS, Snyder SH (1986) Adenosinecontaining neurons in the brain localized by immunocytochemistry. J Neurosci 6:1952–1961Google Scholar
  8. Brandtzaeg P, Rognum TO (1984) Evaluation of nine different fixatives. 2. Preservation of IgG, IgA and secretory component in an artificial immunohistochemical test substrate. Histochemistry 81:213–219Google Scholar
  9. Bras H, Chazal G, Destombes J, Puizillout JJ (1986) Anti-5-hydroxytryptamine antibodies: studies on their cross-reactivity in vitro and their immunohistochemical specificity. Exp Brain Res 63:627–638Google Scholar
  10. Brooker G, Harper JF, Terasaki WL, Moylan RD (1979) Radioimmunoassay of cyclic AMP and cyclic GMP. Adv Cycl Nucl Res 10:1–26Google Scholar
  11. Chan-Palay V, Palay SL (1979) Immunocytochemical localization of cyclic GMP: light and electron microscope evidence for involvement of neuroglia. Proc Natl Acad Sci USA 76:1485–1488Google Scholar
  12. Cumming R, Dickison S, Arbuthnott G (1980) Cyclic nucleotide losses during tissue processing for immunocytochemistry. J Histochem Cytochem 28:54–55Google Scholar
  13. De Vente J, Garssen J, Tilders FJH, Steinbusch HWM, Schipper J (1987a) Single cell quantitative immunocytochemistry of cyclic GMP in the superior cervical ganglion of the rat. Brain Res 411:120–128Google Scholar
  14. De Vente J, Steinbusch HWM, Schipper J (1987b) 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 22:361–373Google Scholar
  15. De Vente J, Bol JGJM, Hudson L, Schipper J, Steinbusch HWM (1988) Atrial natriuretic factor-responding and cyclic guanosine monophosphate (cGMP)-producing cells in the rat hippocampus: a combined micropharmacological and immunocytochemical approach. Brain Res 446:387–395Google Scholar
  16. Friedl A, Harmening C, Schuricht B, Hamprecht B (1985) Rat atrial natriuretic peptide elevates the level of cGMP is astrogliarich brain cell cultures. Eur J Pharmacol 111:141–142Google Scholar
  17. Geysen HM, Tainer JA, Rodda SJ, Mason JJ, Alexander H, Getzoff ED, Lerner RA (1987) Chemistry of antibody binding to a protein. Science 235:1184–1190Google Scholar
  18. Hancock MB (1982) DAB-Nickel substrate for the differential immunoperoxidase staining of nerve fibers and fiber terminals. J Histochem Cytochem 30:578–583Google Scholar
  19. Ignarro LJ, Kadowitz PJ (1985) The pharmacological and physiological role of cGMP in vascular smooth muscle relaxation. Annu Rev Pharmacol Toxicol 25:171–191Google Scholar
  20. Larsson LI (1981) A novel immunocytochemical model-system for specificity and sensitivity screening of antisera against multiple antigens. J Histochem Cytochem 29:408–410Google Scholar
  21. Leitman DC, Murad F (1987) Atrial natriuretic factor receptor heterogeneity and stimulation of particulate guanylate cyclase and cyclic GMP accumulation. Clin Endocrinol Metab 16:79–125Google Scholar
  22. Lowry OH, Rosebrough NJ, Farr AL, Randall FJ (1951) Protein measurement with the Folin reagent. J Biol Chem 193:265–275Google Scholar
  23. Milstein C, Wright B, Cuello AC (1983) The discrepancy between the cross-reactivity of a monoclonal antibody to serotonin and its immunohistochemical specificity. Mol Immunol 20:113–123Google Scholar
  24. Murad F (1986) Cyclic guanosine monophosphate as a mediator of vasodilatation. J Clin Invest 78:1–5Google Scholar
  25. Ortez RA, Sikes RW, Sperling HG (1980) Immunohistochemical localization of cyclic GMP in goldfish retina. J Histochem Cytochem 28:263–270Google Scholar
  26. Petrusz P, Sar M, Ordronneau P, DiMeo P (1976) Specifity of immunocytochemical staining. J Histochem Cytochem 24:1110–1115Google Scholar
  27. Pool CW, Buijs RM, Swaab DF, Boer GJ, Van Leeuwen FW (1983) On the way to a specific immunocytochemical localization. In: Cuello AC (ed) IBRO Handbook series: Methods in the neurosciences, vol. 3. John Wiley, London New York, pp 1–46Google Scholar
  28. Rall TW, Lehne RA (1982) Evidence for crosslinking of cyclic AMP to constituents of brain tissue by aldehyde fixatives: potential utility in histochemical procedures. J Cycl Nucl Res 8:243–265Google Scholar
  29. Schipper J, Tilders FJH (1983) A new technique for studying specificity of immunocytochemical procedures: specificity of serotonin immunostaining. J Histochem Cytochem 31:12–18Google Scholar
  30. Schipper J, Werkman TR, Tilder FJH (1984) Quantitative immunocytochemistry of corticotropin-releasing factor (CRF). Studies on nonbiological models and on hypothalamic tissues of rats after hypophysectomy, adrenalectomy and dexamethasone treatment. Brain Res 293:111–118Google Scholar
  31. Scopsi L, Wang B, Larsson LI (1986) Nonspecific immunocytochemical reaction with certain neurohormonal peptides and basic peptide sequences. J Histochem Cytochem 54:1469–1475Google Scholar
  32. Sokal RR, Rohlf FJ (1969) Biometry. Freeman, San FranciscoGoogle Scholar
  33. Steinbusch HWM, De Vente J, Schipper J (1986) Immunohistochemistry of monoamines in the central nervous system. In: Panula P, Paivrinta H, Soinila S (eds) Neurochemistry. Modern methods and applications. Alan R Liss, New York, pp 75–105Google Scholar
  34. Steinbusch HWM, Wouterlood FG, De Vente J, Bol JGJM, Berkenbosch F (1988) Immunohistochemical localization of monoamines and cyclic nucleotides. Their application in quantitative immunofluorescence studies and tracing monoaminergic neuronal connections. Acta Histochem 35:S85–106Google Scholar
  35. Steiner AL, Parker CW, Kipnis DM (1972) Radioimmunoassay for cyclic nucleotides. 1. Preparation of antibodies and iodinated cyclic nucleotides. J Biol Chem 247:1106–1113Google Scholar
  36. Steiner AL, Ong S, Wedner HJ (1976) Cyclic nucleotide immunocytochemistry. Adv Cycl Nucl Res 7:115–155Google Scholar
  37. Sternberger LA, Hardy PH, Cuculis JH, Meyer HG (1970) The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 18:315–333Google Scholar
  38. Swaab DF, Pool CW, Van Leeuwen FW (1977) Can specificity ever be proved in immunocytochemical staining? J Histochem Cytochem 25:388–390Google Scholar
  39. Tandler CJ, Brusco A, Peressini S, Pecci Saavedra J (1986) Further evidence for the specificity of anti-5-HT (serotonin)-like antisera in immunocytochemistry. Existence of cyclic secondary amino groups in the immunogen. Histochemistry 85:67–72Google Scholar
  40. Tilders FJH, Ploem JS, Smelik PG (1974) Quantitative microfluorimetric studies on formaldehyde-induced fluorescence of 5-hydroxytryptamine in the pineal gland of the rat. J Histochem Cytochem 22:967–975Google Scholar
  41. Van Regenmortel MHV (1987) Antigenic cross-reactivity between proteins and peptides: new insights and applications. Trends Biochem Sci 12:237–240Google Scholar
  42. Waldman SA, Murad F (1987) Cyclic GMP synthesis and function. Pharmacol Rev 39:163–196Google Scholar
  43. Wedner HJ, Hoffer BJ, Battenberg E, Steiner AL, Parker CW, Bloom FE (1972) A method for detecting intracellular cyclic adenosine monophosphate by immunofluorescence. J Histochem Cytochem 20:293–299Google Scholar
  44. Swiller J, Ghandour MS, Revel MO, Basset P (1981) Immunohistochemical localization of guanylate cyclase in rat cerebellum. Neurosci Lett 23:31–36Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • J. de Vente
    • 1
  • J. Schipper
    • 2
  • H. W. M. Steinbusch
    • 1
  1. 1.Department of Pharmacology, Faculty of MedicineFree UniversityAmsterdamThe Netherlands
  2. 2.Department of PharmacologyDUPHAR BVWeespThe Netherlands

Personalised recommendations