11 In Situ Hybridization for RNA: Nonradioactive Probe: Oligo-DNA Probe
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Visualization System
• Direct/indirect enzyme immunohistochemistry
In detail, the following steps have to be performed:
(For specimens prepared with cross-linking fixatives such as formarin.)
Pretreatment steps
1. To inhibit the activity of endogenous peroxidase: Immerse in
0.3-3 % H20 2 in methanol.
2. To remove interfering proteins, especially basic proteins such
as histone and RNase: Treat with 0.2 N HCl.
3. To increase the probe access to target mRNAs in tissues and to
remove cellular matrix proteins: Use of proteinase K.
Hybridization
• Considering of the melting temperature (Tm) : optimal hybridization temperature = Tm - 25°C. Use of formamide (deionized) : Deionized formamide should be used to reduce
Tm. Ten percent (v/v) formamide decreases the Tm by
about 6 °C (Meinkoth 1984).
Stringent washing
• This step for washing is as important as that for pretreatment
steps and hybridization.
Enzyme immunohistochemistry
• Routine direct/indirect enzyme immunohistochemistry is
performed.
Evaluation of results
• To confirm the specificity of target mRNA signals, various
types of control experiments should be conducted concurrently.