11 In Situ Hybridization for RNA: Nonradioactive Probe: Oligo-DNA Probe
165
2. Paraffin embedded sections: warm at 60
0
e (at least 30
mins.), deparaffinize through toluene (4x 5 mins) and
then rehydrate with graded ethanol solutions (5 mins,
100%, 100%,95%,90%,80%,70%).
3. Rinse with PBS (3x 5 mins).
4. Immerse in 0.3% H20 2 in methanol for 15 mins to inhibit
endogenous Streptavidin-binding activity and quenching
peroxidase activity, if necessary.
5. Rinse in PBS (5 mins) and wash with DDW.
6. Immerse in 0.2N Hel for 20 mins and wash successively with
DDW and PBS.
7. Immerse in 0.2% Triton X-lOO in PBS for 10 mins and wash
with PBS.
Note: Triton X-lOO enhances the staining intensity of target
mRNA.
8. Treat with proteinase K (1-100 Ilg/ml in PBS) at 37°e for 15
mins and wash with PBS (3x 5 mins).
Note: The effect of proteinase K digestion varies with the tissue
and type of fixation, so the concentration of proteinase K should
be optimized by individual experiment (To optimize the concentration of proteinase K, we use 28S rRNA probe to determinse the
concentration of proteinase K.). Generally, in fixed frozen sections, maximal staining is obtained at proteinase K Illg/ml, and
in paraffin-embedded sections, maximal staining is obtained at
proteinase K between 10 and 100 Ilg/ml concentrations.
Note: Before this procedure, proteinase K medium is incubated
in a water bath at 37°e for 30 mins.
9. Post-fix the slides with 4% PFA IPBS for 5 mins and wash
with PBS.
10. Immerse in 2 mg/ml glycine/PBS for 15 mins twice and wash
with DDW.
Note: To stop the reaction of 4% PFA/PBS, the slides should be
completely immersed in this medium.
165
2. Paraffin embedded sections: warm at 60
0
e (at least 30
mins.), deparaffinize through toluene (4x 5 mins) and
then rehydrate with graded ethanol solutions (5 mins,
100%, 100%,95%,90%,80%,70%).
3. Rinse with PBS (3x 5 mins).
4. Immerse in 0.3% H20 2 in methanol for 15 mins to inhibit
endogenous Streptavidin-binding activity and quenching
peroxidase activity, if necessary.
5. Rinse in PBS (5 mins) and wash with DDW.
6. Immerse in 0.2N Hel for 20 mins and wash successively with
DDW and PBS.
7. Immerse in 0.2% Triton X-lOO in PBS for 10 mins and wash
with PBS.
Note: Triton X-lOO enhances the staining intensity of target
mRNA.
8. Treat with proteinase K (1-100 Ilg/ml in PBS) at 37°e for 15
mins and wash with PBS (3x 5 mins).
Note: The effect of proteinase K digestion varies with the tissue
and type of fixation, so the concentration of proteinase K should
be optimized by individual experiment (To optimize the concentration of proteinase K, we use 28S rRNA probe to determinse the
concentration of proteinase K.). Generally, in fixed frozen sections, maximal staining is obtained at proteinase K Illg/ml, and
in paraffin-embedded sections, maximal staining is obtained at
proteinase K between 10 and 100 Ilg/ml concentrations.
Note: Before this procedure, proteinase K medium is incubated
in a water bath at 37°e for 30 mins.
9. Post-fix the slides with 4% PFA IPBS for 5 mins and wash
with PBS.
10. Immerse in 2 mg/ml glycine/PBS for 15 mins twice and wash
with DDW.
Note: To stop the reaction of 4% PFA/PBS, the slides should be
completely immersed in this medium.