Summary
This chapter describes a pre-embedding in situ hybridization method utilizing an immunogold-silver intensification step to identify P2Y2 receptor mRNA transcripts in the adult rat cerebellum. The method was applied for ultrastructural (electron microscopic) examination. Transcripts for P2Y2 receptors, marked by the gold-silver grains, were revealed in Purkinje cells. Transcripts were essentially localized in the cytoplasm although they also appeared to be specifically associated with granular endoplasmic reticulum. This suggests that Purkinje cells may produce functional P2Y2 receptors involved in the ATP-related regulatory role in the cerebellum. The in situ hybridization method that was applied enabled simultaneous preservation of tissue ultrastructure and localization of labeled mRNA transcripts. This method may be adapted and used to study various tissues.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Glass R., Townsend-Nicholson A., and Burnstock G. (2000) P2 receptors in the thymus: expression of P2X and P2Y receptors in adult rats, an immunohistochemical and in situ hybridisation study. Cell Tissue Res. 300, 295–306.
Matsuno A., Nagashima T., Ohsugi Y., et al. (2000) Electron microscopic observation of intracellular expression of mRNA and its protein product: technical review on ultrastructural in situ hybridization and its combination with immunohistochemistry. Histol. Histopathol. 15, 261–268.
Kloc M., Bilinski S., Pui-Yee C. A., and Etkin L. D. (2000) The targeting of Xcat2 mRNA to the germinal granules depends on a cis-acting germinal granule localization element within the 3’UTR. Dev. Biol. 217, 221–229.
Loesch A., Glass R., and Burnstock G. (2002) Ultrastructural identification of P2Y2 receptor mRNA in the rat thymus. Cell Tissues Organs 172, 255–264.
Burnstock G. (1978) A basis for distinguishing two types of purinergic receptors, in Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach (Straub R. W. and Bolis L., eds.), Raven Press, New York, pp. 107–118.
Abbracchio M. P. and Burnstock G (1998) Purinergic signalling: pathophysiological roles. Jpn. J. Pharmacol. 78, 113–145.
Ralevic V. and Burnstock G. (1998) Receptors for purines and pyrimidines. Pharmacol. Rev. 50, 413–492.
Loesch A. and Burnstock G. (1998) Electron-immunocytochemical localization of P2X1 receptors in the rat cerebellum. Cell Tissue Res. 294, 253–260.
Loesch A. and Burnstock G. (2000) Ultrastructural localisation of ATP-gated P2X2 receptor immunoreactivity in vascular endothelial cells in rat brain. Endothelium 7, 93–98.
Loesch A. and Burnstock G. (2001) Immunoreactivity to P2X6 receptors in the rat hypothalamo-neurohypophysial system: an ultrastructural study with ExtrAvidin and colloidal gold-silver labeling. Neuroscience 106, 621–631.
Loesch A., Miah S., and Burnstock G. (1999) Ultrastructural localization of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamo-neurohypophysial system. J. Neurocytol. 28, 495–504.
Loesch A. (2002) Perivascular nerves and vascular endothelium: recent advances. Histol. Histopathol. 17, 591–597.
Hayat M. A. (ed.) (1995) Immunogold-Silver Staining. Principle, Methods, and Applications. CRC Press, Boca Raton, FL.
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Loesch, A., Glass, R. (2006). Electron Microscopy and In Situ Hybridization. In: Darby, I.A., Hewitson, T.D. (eds) In Situ Hybridization Protocols. Methods in Molecular Biology™, vol 326. Humana Press. https://doi.org/10.1385/1-59745-007-3:151
Download citation
DOI: https://doi.org/10.1385/1-59745-007-3:151
Publisher Name: Humana Press
Print ISBN: 978-1-58829-402-9
Online ISBN: 978-1-59745-007-2
eBook Packages: Springer Protocols