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YOSHITAKA HISHIKAWA ET AL.
Before starting the procedure of in situ hybridization with T -T
dimer method on tissue sections, it is necessary to label the oligonucleotide with T -T dimer and then to check the labeled probe
by immunohistochemical detection and the hybridizablity by
dot blot hybridization on a nitrocellulose filter respectively.
Outline
• T -T dimerization of DNA
• Immunohistochemical detection ofT-T dimerized DNA on a
nitrocellulose filter
• Dot blot hybridization with T -T dimerized DNA probes
• In situ hybridization with T -T dimerized probes
A short protocol of the entire procedure is shown in the following:
Pretreatment Steps
• Inhibiting the activity of endogenous peroxidase
• Removing proteins such as histone and cellular matrix proteins
• Post-fixation of the specimens
Hybridization
• Base-pairing between target nucleic acids in cells or tissues
and probe nucleic acids
Stringent Washing
• Reducing the level of background staining and bringing about
an increase in the signal/noise ratio