Abstract
Section in situ hybridization using either radioactive or nonradioactive labeled cDNA probes is an invaluable technique that enables the investigator to detect and localize mRNA expression within tissue sections and cells. Here, we describe the labeling of 35S-UTP radioactive and nonradioactive digoxigenin probes, preparation of tissue sections, hybridization, and washing of non-hybridized probes, followed by the detection of radioactive signals via dipping in nuclear emulsion and the immunohistochemical and subsequent colorimetric detection of nonradioactive signals.
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References
Gall JG, Pardue ML (1969) Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci U S A 63(2):378–383
Angerer LM, Angerer RC (1981) Detection of poly A + RNA in sea urchin eggs and embryos by quantitative in situ hybridization. Nucleic Acids Res 9(12):2819–2840
Wilcox JN (1993) Fundamental principles of in situ hybridization. J Histochem Cytochem 41(12):1725–1733
Conway SJ (1996) In situ hybridization of cells and tissue sections. Methods Mol Med 6:193–206
Micales BK, Lyons GE (2001) In situ hybridization: use of 35S-labeled probes on paraffin tissue sections. Methods 23(4):313–323
Liu CQ, Shan L, Balesar R, Luchetti S, Van Heerikhuize JJ, Luo JH, Swaab DF, Bao AM (2010) A quantitative in situ hybridization protocol for formalin-fixed paraffin-embedded archival post-mortem human brain tissue. Methods 52(4):359–366
Winzer-Serhan UH, Broide RS, Chen Y, Leslie FM (1999) Highly sensitive radioactive in situ hybridization using full length hydrolyzed riboprobes to detect alpha 2 adrenoceptor subtype mRNAs in adult and developing rat brain. Brain Res Brain Res Protoc 3(3):229–241
Nuovo GJ (2010) In situ detection of microRNAs in paraffin embedded, formalin fixed tissues and the co-localization of their putative targets. Methods 52(4):307–315
Cloëz-Tayarani I, Fillion G (1997) The in situ hybridization and immunocytochemistry techniques for characterization of cells expressing specific mRNAs in paraffin-embedded brains. Brain Res Brain Res Protoc 1(2):195–202
Toledano H, D’Alterio C, Loza-Coll M, Jones DL (2012) Dual fluorescence detection of protein and RNA in Drosophila tissues. Nat Protoc 7(10):1808–1817
Koushik SV, Bundy J, Conway SJ (1999) Sodium-calcium exchanger is initially expressed in a heart-restricted pattern within the early mouse embryo. Mech Dev 88(1):119–122
Snider P, Hinton RB, Moreno-Rodriguez RA, Wang J, Rogers R, Lindsley A, Li F, Ingram DA, Menick D, Field L, Firulli A, Molkentin JD, Markwald R, Conway SJ (2008) Periostin is required for maturation and extracellular matrix stabilization of noncardiomyocyte lineages of the heart. Circ Res 102(7):752–760
Acknowledgements
This work was supported, in part, by American Heart Association 12PRE9430047 Predoctoral Fellowship (OS); as well as the Riley Children’s Foundation, the Indiana University Department of Pediatrics (Neonatal–Perinatal Medicine) and NIH HL60714 grant (SJC).
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Simmons, O., Bolanis, E.M., Wang, J., Conway, S.J. (2014). In Situ Hybridization (Both Radioactive and Nonradioactive) and Spatiotemporal Gene Expression Analysis. In: Singh, S., Coppola, V. (eds) Mouse Genetics. Methods in Molecular Biology, vol 1194. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1215-5_12
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DOI: https://doi.org/10.1007/978-1-4939-1215-5_12
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