Abstract
In situ hybridization is a powerful tool for visualizing cellular gene expression in morphologically preserved brain tissue giving precise information on the regional expression of specific mRNA sequences in cells of diverse phenotype. Here, we describe a sensitive, simple, and robust method using alkaline phosphatase (AP)-labelled oligodeoxynucleotide probes to detect cytokine mRNA in the acutely injured or inflamed mouse CNS.
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References
Gregersen R, Lambertsen KL, Finsen B (2000) Microglia and macrophages are major sources of tumor necrosis factor in permanent middle cerebral occlusion in mice. J Cereb Blood Flow Metab 20:53–65
Clausen BH, Lambertsen KL, Meldgaard M et al (2005) A quantitative in situ hybridization and polymerase chain reaction study of microglial-macrophage expression of interleukin-1beta mRNA following permanent middle cerebral artery occlusion in mice. Neuroscience 132:879–892
Lambertsen KL, Clausen BH, Babcock AA et al (2009) Microglia protect neurons against ischemia by synthesis of tumor necrosis factor. J Neurosci 29:1319–1330
Christensen JE, Simonsen S, Fenger C et al (2009) Fulminant lymphocytic choriomeningitis virus-induced inflammation of the CNS involves a cytokine-chemokine-cytokine-chemokine cascade. J Immunol 182:1079–1087
Buttini M, Appen K, Sauter A et al (1996) Expression of tumor necrosis factor alpha after focal cerebral ischemia in the rat. Neuroscience 71:1–16
Meltzer JC, Sanders V, Grimm PC et al (1998) Production of digoxigenin-labelled RNA probes and the detection of cytokine mRNA in rat spleen and brain by in situ hybridization. Brain Res Prot 2:339–351
Woodroofe MN, Cuzner ML (1993) Cytokine mRNA expression in inflammatory multiple sclerosis lesion: detection by non-radioactive in situ hybridization. Cytokine 5:583–588
Kiefer R, Funa K, Schweitzer T et al (1996) Transforming growth factor-beta 1 in experimental autoimmune neuritis. Cellular localization and time course. Am J Pathol 148:211–223
Turrin NP, Rivest S (2006) Tumor necrosis factor a but not interleukin 1β mediates neuroprotection in response to acute nitric oxide excitotoxicity. J Neurosci 26:143–151
Höfler H, Childers H, Montminy MR et al (1986) In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes. Histochem J 18:597–604
VandenBroecke C, Tovey MG (1991) Expression of the genes of class I interferons and interleukin-6 in individual cells. J Interferon Res 11:91–103
Lu J, Tsourkas A (2009) Imaging individual microRNAs in single mammalian cells in situ. Nucleic Acids Res 37:e100
Tecott LH, Eberwine JH, Barchas JD et al (1987) Methodological considerations in the utilization of in situ hybridization. In: Eberwine JH, Barchas JD, Valentino KL (eds) In situ hybridization: amplification to neurobiology. Oxford Press, New York, NY, pp 3–24
Finsen B, Gregersen R, Lehrmann E et al (2004) In situ hybridization. In: Evans SM, Janson AM, Nyengaard JR (eds) Quantitative methods in neuroscience—a neuroanatomical approach. Oxford University Press, New York, NY, pp 115–145
Clausen BH, Lambertsen KL, Finsen B (2006) Glyceraldehyde-3-phosphate dehydrogenase versus toluidine blue as a marker for infarct volume estimation following permanent middle cerebral artery occlusion in mice. Exp Brain Res 175:60–67
Fenger C, Drøjdahl N, Wirenfeldt M et al (2006) Tumor necrosis factor or its TNFp55 and -p75 receptors are not required for axonal lesion-induced microgliosis in mouse fascia dentata. Glia 54:591–605
Pennica D, Hayflick JS, Bringman TS et al (1985) Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor. Proc Natl Acad Sci USA 82:6060–6064
Gray BW, Glaister D, Chen E et al (1986) Two interleukin 1 genes in the mouse: cloning and expression of the cDNA for murine interleukin-1b. J Immunol 137:3644–3648
Sabath DE, Broome HE, Prytowsky MB (1990) Glyceraldehyde-3-phosphate dehydrogenase mRNA is a major interleukin-2-induced transcript in a cloned T-helper lymphocyte. Gene 91:185–191
Acknowledgment
Lene Jørgensen and Sussanne Petersen are acknowledged for their excellent technical assistance. The experimental material shown in Fig. 1 was kindly provided by Dr. Kate Lambertsen. The work was supported by grants from the Lundbeck Foundation, the Novo Nordisk Foundation, and the Danish Multiple Sclerosis Society.
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Clausen, B., Fenger, C., Finsen, B. (2013). In Situ Hybridization of Cytokine mRNA Using Alkaline Phosphatase-Labelled Oligodeoxynucleotide Probes. In: Joseph, B., Venero, J. (eds) Microglia. Methods in Molecular Biology, vol 1041. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-520-0_10
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DOI: https://doi.org/10.1007/978-1-62703-520-0_10
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