Abstract
Immunocytochemistry (ICC) of small, water-soluble molecules requires some special precautions compared to ICC of large protein molecules. In general, protein antigens are relatively insensitive to the type of fixative used. Apparently, the epitopes are not altered in a decisive way by the fixative, immobilization is usually effective, and more than one eptiope may be recognized by polyclonal antisera. In studying by ICC the location of small, hydrophilic molecules—such as neurotransmitters and cyclic nucleotides—choice of fixative plays a decisive role. First, fixation is necessary not only to preserve tissue morphology, but also to fix the molecules to the tissue matrix, otherwise these may be lost from the tissue. This is certainly the case for cyclic nucleotides (1–4). Second, the fixative may covalently modify the hapten by fixing it to tissue protein. This has been shown to occur with serotonim (5–6) and also with small peptides (7). Because the covalent modification of the hapten may be part of the antigenic determinant, it is a prerequisite that antisera are raised against conjugates prepared using a fixative that is also suitable for tissue fixation (8–10).
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References
Cumming, R., Dickison, S., and Arbuthnott, G. (1980) Cyclic nucleotide losses during tissue processing for immunocytochemistry. J. Histochem. Cytochem. 28, 55.
Ortez, R. A., Sikes, R. W., and Sperling, H. G. (1980) Immunohistochemical localization of cyclic GMP in goldfish retina. J. Histochem. Cytochem. 28, 263–270.
Rall, T. W. and Lehne, R. A. (1982) Evidence for cross-linking of cyclic AMP to constituents of brain tissue by aldehyde fixatives: potential utility in histochemical procedures. J. Cycl. Nucleotides Res. 8, 243–265.
Steiner, A. L., Ong, S., and Wedner, H. J. (1976) Cychc nucleotide immunocytochemistry. Adv. Cycl. Nucleotides Res. 7, 115–155.
Schipper, J. and Tilders, F. J. H. (1983) A new technique for studying specificity of immunocytochemical procedures: specificity of serotonin immunostaining. J. Histochem. Cytochem. 31, 12–18.
Milstein, C., Wright, B., and Cuello, A. C. (1983) The discrepancy between the cross-reactivity of a monoclonal antibody to serotonin and its immunohistochemical specificity. Mol. Immunol. 20, 113–123.
Berkenbosch, F., Schipper, J., and Tilders, F. J. H. (1986) Corticotropin-releasing factor immunostaining in the rat spinal cord and medulla oblongata: an unexpected form of cross-reactivity with substance P. Brain Res. 399, 87–96.
De Vente, J., Steinbusch, H. W. M., and Schipper, J. (1987) 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, 361–373.
De Vente, J., Schipper, J., and Steinbusch, H. W. M. (1989) Formaldehyde fixation of cGMP in distinct cellular pools and then recognition by different cGMP-antisera. An immunocytochemical study into the problem of serum specificity. Histochemistry 91, 401–412.
De Vente, J. and Steinbusch, H. W. M. (1992) On the stimulatron of soluble and particulate guanylate cyclase in the rat brain and the involvement of nitric oxide as studied by cGMP-immunocytochemistry. Acta. Histochem. 92, 13–38.
Steiner, A. L., Parker, C. W., and Kipnis, D. M. (1972) Radioimmunoassay for cyclic nucleotides. 1. Preparation of antibodies and iodinated cyclic nucleotides. J. Biol. Chem. 247, 1106–1113.
Rosenberg, E. M., LaVallee, H., Weber, P., and TUCCI, S. M. (1979) Studies on the specificity of immunohistochemical techniques for cyclic AMP and cyclic GMP. J. Histochem. Cytochem. 27, 913–923.
Wedner, H. J., Hoffer, B. J., Battenberg, E., Steiner, A. L., Parker, C. W., and Bloom, F. E. (1972) A method for detecting intracellular cyclic adenosine monophosphate by immunofluorescence. J. Histochem. Cytochem. 20, 293–299.
Chan-Palay, V. and Palay, S. L. (1979) Immunohistochemical localization of cyclic GMP: light and electron microscope evidence for involvement of neuroglia. Proc. Natl. Acad. Sci. USA 76, 1485–1488.
Garbers, D. L. and Lowe, D. G. (1994) Guanylyl cyclase receptors. J. Biol. Chem. 269, 30,741–30,744.
Waldman, S. A. and Murad, F. (1987) Cyclic GMP synthesis and function. Pharmacol. Rev. 39, 163–196.
Murad, F. (1994) Regulation of cytosolic guanylyl cyclase by nitric oxide: the NO-cyclic GMP signal transduction system. Adv Pharmacol. 26, 19–33.
Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1991) Nitric oxide. physiology, pathophysiology and pharmacology. Pharmacol. Rev. 43, 109–142.
Garthwaite, J. (1991) Glutamate, nitric oxide and cell-cell signalling in the nervous system. Trends Neurosa. 14, 60–67.
Knowles, R. G. and Moncada, S. (1994) Nitric oxide synthases in mammals. Biochem. J. 298, 249–258.
Palmer, R. M. J., Ashton, D. S., and Moncada, S. (1988) Vascular endothelial cells synthesize nitric oxide from L-argirine. Nature 333, 664–666.
Marletta, M. A. (1993) Nitric oxide synthase. structure and mechanism. J. Biol. Chem. 268, 12231–12234.
Bredt, D. S., Hwang, P. M., and Snyder, S. H. (1990) Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347, 768–770.
Vincent, S. R. and Kimura, H. (1992) Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 46, 755–784.
Rodrigo, J., Springall, D. R., Uttenthal, O., Bentura, M. L., Abadia-Molma, F., Riveros-Moreno, V., Martinez-Murillo, R., Polak, J. M., and Moncada, S. (1994) Localization of nitric oxide synthase in the adult rat brain. Phil. Trans. R. Soc. Lond. B. 345, 175–221.
Knowles, R. G., Palacios, M., Palmer, R. M. J., and Moncada, S. (1989) Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc. Natl. Acad. Sci. USA 86, 5159–5162.
Bredt, D. S. and Snyder, S. H. (1992) Nitric oxide, a novel neuronal messenger. Neuron. 8, 3–11.
De Vente, J., Bol., J. G. J. M., Berkelmans, H. S., Schipper, J., and Steinbusch, H. W. M. (1990) Immunocytochemistry of cyclic GMP in the cerebellum of the immature, adult, and aged rat: the involvement of nitric oxide. A micro-pharmacological study. Eur. J. Neurosci. 2, 845–862.
De Vente, J. and Steinbusch, H. W. M. (1993) Immunocytochemistry of second messenger molecules. the study of formaldehyde-fixed cyclic GMP, in Immunohutochemistry II (Cuello, A. C., ed.), Wiley, New York, pp. 409–427.
Southam, E. and Garthwaite, J. (1993) The nitric oxide-cyclic GMP signalling pathway in rat brain. Neuropharmacology 32, 1267–1277.
Koistinaho, J., Swanson, R. A., De Vente, J., and Sagar, S. M. (1993) NADPH-diaphorase (nitric oxide synthase)-reactive amacrine cells of rabbit retina: putative target cells and stimulation by light. Neuroscience 57, 587–597.
Young, H. M., McConalogue, K., Furness, J. B., and De Vente, J. (1993) Nitric oxide targets in the guinea-pig intestine identified by induction of cyclic GMP immunoreactivity. Neuroscience 55, 583–596.
Shuttleworth, C. W., Xue, C., Ward, S. M., De Vente, J., and Sanders, K. M. (1993) Immunohistochemical localization of 3′,5′-cyclic guanosine monophosphate in the canine proximal colon. responses to nitric oxide and electrical stimulation of enteric inhibitory neurons. Neuroscience 56, 513–522.
De Vente, J., Schipper, J., and Steinbusch, H. W. M. (1993) A new approach to the immunocytochemistry of cAMP. Initial characterization of antibodies against acrolein-fixed cAMP. Histochemistty 99, 457–462.
Berkenbosch, F. and Tilders, F. J. H. (1987) A quantitative approach to cross-reaction problems in immunocytochemistry. Neuroscience 23, 823–826.
Larsson, L. I. (1981) A novel immunocytochemical model-system for specificity and sensitivity screening of antisera against multiple antigens. J. Histochem. Cytochem. 29, 408–410.
De Vente, J., Garssen, J., Tilders, F. J. H., Steinbusch, H. W. M., and Schipper, J. (1987) Single cell quantitative immunocytochemistry of cyclic GMP in the superior cervical ganglion of the rat. Brain Res. 411, 120–128.
Atassi, M. Z. (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, 1–20.
Kant, G. J., Muller, T. W., Lenox, R. H., and Meyerhoff, J. L. ( 1982) In vivo effects of pentobarbital and halothane anesthesia in levels of adenosine 3′,5′-monophosphate and guanosine 3′, 5′-monophosphate in rat brain regions and pituitary. Biochem. Pharmacol. 29, 1891–1896.
Garthwaite, J., Woodham, P. L., Collins, M. J., and Balasz, R. (1979) On the preparation of brain slices: morphology and cyclic nucleotides. Brain Res. 173, 373–377.
Aitken, P. G., Breese, G. R., Dudek, F. F., Edwards, F., Espanol, M. T., Larkman, P. M., Lipton, P., Newman, G. C., Nowak, T. S., Panizzon, K. L., Raly-Susman, K. M., Reid, K. H., Rice, M. E., Sarvey, J. M., Schoepp, D. D., Segal, M., Taylor, C. P., Teyler, T. J., and Voulalas, P. J. (1995) Preparative methods for brain slices. a discussion. J. Neurosci. Meth. 59, 139–149.
Lipinski, H. G. and Bingmann, D. (1986) pO2-dependent distribution of potassium in hippocampal slices of the guinea pig. Brain Res. 380, 267–275.
Lipinski, H. G. and Bingmann, D. (1987) Diffusion in slice preparations bathed in unstirred solutions. Brain Res. 437, 26–34.
De Vente, J., Bol, J. G. J. M., and Steinbusch, H. W. M. (1989) cGMP-producing, atrial natriuretic factor-responding cells in the rat brain. An immunocytochemical study. Eur. J. Neurosci. 1, 436–460.
Wood, J. and Garthwaite J. (1994) Models of the diffusional spread of nitric oxide: implications for neural nitric oxide signalling and its pharmacological properties. Neuropharmacology 33, 1235–1244.
East, S. J., Batchelor, A. M., and Garthwaite, J. (1991) Selective blockade of N-methyl-D-aspartate receptor function by the nitric oxide donor, nitroprusside. Eur. J. Pharmacol. 209, 119–121.
Sonnenburg, W. K. and Beavo, J. A. (1994) Cyclic GMP and regulation of cyclic nucleotide hydrolysis. Adv. Pharmacol. 26, 87–114.
Beavo, J. A. and Reifsnyder, D. H. (1990) Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors. Trends Neurosci. 11, 150–155.
Billingsley, M. L., Polli, J. W., Balaban, C. D., and Kincaid, R. L. (1990) Developmental expression of calmodulin-dependent cyclic nucleotide phosphodiesterase in rat brain. Dev. Brain Res. 53, 253–263.
Furuyama, T., Iwahashi, Y., Tano, Y, Takagi, H., and Inagaki, S. (1994) Localization of 63-kDa calmodulin stimulated phosphodiesterase mRNA in the rat brain by in situ hybridization histochemistry. Mol. Brain Res. 26, 331–336.
Polli, J. W. and Kincaid, R. L. (1994) Expression of a calmodulin-dependent phosphodiesterase isoform (PDE1B1) correlates with brain regions having extensive dopaminergic innervation. J. Neurosci. 14, 1251–1261.
Vallebuona, F. and Raiteri, M. (1993) Monitoring of cyclic GMP during cerebellar microdialysis in freely-moving rats as an index of nitric oxide synthase activity. Neuroscience 57, 577–585.
Luo, D., Leung, E., and Vincent, S. R. (1994) Nitric-oxide dependent efflux of cGMP in rat cerebellar cortex: an in vivo microdialysis study. J. Neurosci. 14, 263–271.
Laitinen, J. T., Laitinen, K. S. M., Tuomisto, L., and Airaksinen, M. M. (1994) Differential regulation of cyclic GMP levels in the frontal cortex and the cerebellum of anesthetized rats by nitric oxide: an in vivo microdialysis study. Brain Res. 668, 117–121.
Schini, V., Grant, N. J., Miller, R. C., and Takeda, K. (1989) Morphological characterization of cultured bovine aortic endothelial cells and the effects of atriopeptin II and sodium nitroprusside on cellular and extracellular accumulation of cyclic GMP. Eur. J. Cell. Brol. 47, 53–61.
Ørbo, A., Jaeger, R., and Sager, G. (1993) Effect of serum and cell density on transmembrane distribution of cAMP and cGMP in transformed (C4-I1) and nontransformed (WI-38) human cells. Int. J. Cancer 55, 957–962.
Wu, X. B., Brüne, B., Von Appen, F, and Ullrich, V. (1993) Efflux of cyclic GMP from activated human platelets. Mol. Pharmacol. 43, 564–568.
Tjornhammar, M. L., Lazaridis, G., and Bartfai, T. (1986) Efflux of cyclic guanosine 3′,5′-monophosphate from cerebellar slices stimulated by L-glutamate or high K+ or N-methyl-N′-nitro-N-nitrosoguanidine. Neurosci. Lett. 68, 95–99.
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de Vente, J., Steinbusch, H.W.M. (1997). cGMP-Immunocytochemistry. In: Rayne, R.C. (eds) Neurotransmitter Methods. Methods in Molecular Biology, vol 72. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-394-5:125
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DOI: https://doi.org/10.1385/0-89603-394-5:125
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