Abstract
Immunohistochemistry has been used widely for the detection of proteins in brain tissue. The process can be performed on free-floating sections, but thicker sections are required than those required for processing on slides due to the “wear and tear” of the constant agitation that free-floating sections undergo. Immunohistochemical detection of molecules of interest such as receptors, neurotransmitters or intracellular signaling molecules is used to determine the distribution of these molecules in tissues. However, it is often of interest to simultaneously determine where the neurons under investigation may project and whether they are activated by a specific stimulus. In this chapter, we will focus on protocols that we use to combine the detection of (i) Fos-positive neurons to detect increased neuronal activity, (ii) nicotine adenine dinucleotide phosphate-diaphorase (NADPH-d) to detect nitric oxide synthase, and (iii) retrogradely transported tracers to identify specific projections.
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
Sagar, S.M., Sharp, F.R., and Curran, T. (1988) Expression of C-fos protein in the brain: metabolic mapping at the cellular level. Science 240, 1328–1331.
Hughes, P., Lawlor, P., and Dragunow, M. (1992) Basal expression of Fos, Fos-related, Jun, and Krox 24 proteins in rat hippocampus. Mol. Brain Res. 13, 355–357.
Dragunow, M. and Faull, R. (1989) The use of c-fos as a metabolic marker in neuronal pathway tracing. J. Neurosci. Methods 29, 261–265.
Hughes, P. and Dragunow, M. (1995) Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system. Pharmacol. Rev. 47, 133–178.
Curran, T., Abate, C., Baker, S., Kerppola, T., and Xanthoudakis, S. (1993) The regulation of c-fos – too much is never enough. Adv. Second Messenger Phosphoprotein Res. 28, 271–277.
Kerppola, T. and Curran, T. (1995) Transcription – Zen and the art of Fos and Jun. Nature 373, 199–200.
Curran, T. and Morgan, J.I. (1995) Fos: an immediate-early transcription factor in neurons. J. Neurobiol. 26, 403–412.
Smeyne, R.J., Vendrell, M., Hayward, M., Baker, S.J., Miao, G.G., Schilling, K., Robertson, L.M., Curran, T., and Morgan, J.I. (1993) Continuous c-fos expression precedes programmed cell death in vivo. Nature 363, 166–169.
Morgan, J.I. and Curran, T. (1989) Stimulus-transcription coupling in neurons: role of cellular immediate-early genes. Trends Neurosci. 12, 459–462.
Morgan, J.I. and Curran, T. (1991) Proto-oncogene transcription factors and epilepsy. Trends Pharmacol. Sci. 12, 343–349.
Morgan, J.I. and Curran, T. (1986) Role of ion influx in the control of c-fos expression. Nature 322, 552–555.
Badoer, E., McKinley, M.J., Oldfield, B.J., and McAllen, R.M. (1992) Distribution of hypothalamic, medullary and lamina terminalis neurons expressing Fos after hemorrhage in conscious rats. Brain Res. 582, 323–328.
Badoer, E., McKinley, M.J., Oldfield, B.J., and McAllen, R.M. (1993) A comparison of non-hypotensive and hypotensive hemorrhage on Fos expression in spinally-projecting neurons of the paraventricular nucleus and rostral ventrolateral medulla. Brain Res. 610, 216–223.
Badoer, E. (1994) The cardiovascular role of the paraventricular nucleus. Proc. Aust. Physiol. Pharmacol. Soc. 25, 20–25.
Badoer, E., Oldfield, B.J., and McKinley, M.J. (1993) Haemorrhage-induced production of Fos in neurons of the lamina terminalis: role of endogenous angiotensin II. Neurosci. Lett. 159, 151–154.
Badoer, E., McKinley, M.J., Oldfield, B.J., and McAllen, R.M. (1994) Localization of barosensitive neurons in the caudal ventrolateral medulla which project to the rostral ventrolateral medulla. Brain Res. 657, 258–268.
Oldfield, B.J., Badoer, E., Hards, D.K., and McKinley, M.J. (1994) Fos production in retrogradely labeled neurons of the lamina terminalis following intravenous infusion of either hypertonic saline or angiotensin II. Neuroscience 60, 255–262.
McKinley, M.J., Badoer, E., Vivas, L., and Oldfield, B.J. (1995) Comparison of c-fos expression in the lamina terminalis of conscious rats after intravenous or intracerebroventricular angiotensin. Brain Res. Bull. 37, 131–137.
Badoer, E., McKinlay, D., Trigg, L., and McGrath, B.P. (1997) Distribution of activated neurons in the rabbit brain following a volume load. Neuroscience 81, 1065–1077.
Badoer, E. and McKinlay, D. (1997) Effect of intravenous angiotensin II on Fos distribution and drinking behaviour in the rabbit. Am. J. Physiol. 272, R1515–R1524.
Badoer, E. and Merolli, J. (1998) Neurons in the hypothalamic paraventricular nucleus that project to the rostral ventrolateral medulla are activated by haemorrhage. Brain Res. 791, 317–320.
Shafton, A.D., Ryan, A.R., and Badoer, E. (1998) Neurons in the hypothalamic paraventricular nucleus send collaterals to the spinal cord and to the rostral ventrolateral medulla in the rat. Brain Res. 801, 239–243.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Badoer, E. (2010). Retrogradely Transported Neuronal Tracers Combined with Immunohistochemistry Using Free-Floating Brain Sections. In: Hewitson, T., Darby, I. (eds) Histology Protocols. Methods in Molecular Biology, vol 611. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-345-9_6
Download citation
DOI: https://doi.org/10.1007/978-1-60327-345-9_6
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-344-2
Online ISBN: 978-1-60327-345-9
eBook Packages: Springer Protocols