In situ hybridization of synovial tissue.
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 degrees 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.
Kuchen
Stefan
S
Seemayer
Christian A
CA
Neidhart
Michel
M
Gay
Renate E
RE
Gay
Steffen
S
D005629:Frozen Sections; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods*; D007202:Indicators and Reagents; D015335:Molecular Probes; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011868:Radioisotopes; D012260:Ribonucleases; D012996:Solutions; D013583:Synovial Membrane / Q000378:metabolism*
D007202:Indicators and Reagents; D015335:Molecular Probes; D012333:RNA, Messenger; D011868:Radioisotopes; D012996:Solutions; D012260:Ribonucleases