Abstract
The chapter focuses on the detection of specific mRNA by in situ hybridization (ISH) in synovial tissue specimens. This technique is widely applied, reliable, specific, and sensitive, because even small quantities of mRNA can be detected. Presented here contemporary protocols for ISH using a combined nonradioactive immunohistochemical detection system.
In overview, the following steps have to be covered to perform ISH. (1) mRNA probes (sense and antisense) are generated by in vitro transcription of cDNA utilizing digoxigenin-labeled UTP nucleotides, (2) fixed tissue sections are digested with trypsin and treated consecutively with prehybridization solutions, (3) hybridization with labeled riboprobes takes place at 50°C overnight in a humid chamber, (4) unbound riboprobe is removed by incubation with RNase A and additional washing with buffers, (5) stringent washing steps are performed with solutions of different sodium dodecyl sulfate, SSC, and formamide concentrations, (6) digoxigenin-labeled probes are detected immunohistochemically using antidigoxigenin antibodies linked with alkaline phosphatase and NBT/BCIP as detection system.
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References
Schwarzacher, T. and Heslop-Harrison, P. (2000) Practical in Situ Hybridization, Gerland Science (Bios Scientific Publisher).
Beesley, J. E. (2000) Immunocytochemistry and in Situ Hybridization in the Biomedical Sciences, first ed. Birkhauser, Boston Basel Berlin.
Darby, Ian, A. (ed.) (2000) In Situ Hybridization Protocols (Series: Methods in molecular biology), second ed., Humana Press, Totowa, New Jersey.
Wilkinson, D. G. (ed.) (1999) In Situ Hybridization: A Practical Approach, second ed., Oxford University Press, New York.
Morel, G. and Cavalier, A. (2000) In Situ Hybridization in Light Microscopy (Series: Methods of Visualization), CRC Press, Boca Raton Florida.
Online: In situ hybridization and immunohistochemistry (Chapter 14 of Current protocols in molecular biology), Current protocols online, 2003 Wiley and Sons.
Kriegsmann, J, Keyser, G., Geiler, T., Gay, R. E., and Gay, S. (1994) A new double labeling technique for combined in situ hybridization and immunohistochemical analysis. Lab Invest. 71, 911–917.
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Kuchen, S., Seemayer, C.A., Neidhart, M., Gay, R.E., Gay, S. (2007). In Situ Hybridization of Synovial Tissue. In: Cope, A.P. (eds) Arthritis Research. Methods in Molecular Medicine, vol 135. Humana Press. https://doi.org/10.1007/978-1-59745-401-8_4
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DOI: https://doi.org/10.1007/978-1-59745-401-8_4
Publisher Name: Humana Press
Print ISBN: 978-1-58829-344-2
Online ISBN: 978-1-59745-401-8
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