14 In Situ Hybridization for RNA: Nonradioactive Probe: RNA Probe
199
PBS for about 2 hrs, embedded in an embedding substance (for
example O.C.T. compound), and frozen in dry ice/acetone.
Cut sections, mount them on silane-coated glass slides.
If this fixative is used, there are no significant differences of
sensitivity between paraffin-embedded sections and that of frozen sections.
Hybridization (paraffin-embedded sections)
1. Deparaffinize sections in xylene-ethanol series.
2. Heat the sections in lOmM citrate buffer (pH 6.0) by microwave (IS mins after boiling).
3. Wash the sections in PBS.
4. Treat the sections with proteinase K (20)lg/ml in PBS) for 30
mins at 37°C.
5. Wash the sections in PBS, 3x.
6. Fix the sections in 4% PFA in PBS for 5 mins.
7. Immerse the sections in glycine (IOmg/ml) for 15 mins, 2x.
8. Immerse the sections in 2x SSC/50% deionized formamide,
30mins
The procedure step 6-8 can be replaced by dehydration by
ethanol and dry-up. The heating procedure (step 2) is used to
enhance the sensitivity (mildly effective).
For frozen sections, first cut frozen sections, mount them
onto coated-glass slides, and air-dry them for Ihr. When fresh
frozen sections are used, fix tissue sections with 4% PF A for 30
mins. Immerse sections in PBS to remove OCT compound. The
hybridization procedures are the same as those of paraffin-embedded sections except that the condition of proteinase K treatment is mild; i.e., 2)lg/ml for 10 mins at 37°C.
Prehybridization (RNasefree condition)
199
PBS for about 2 hrs, embedded in an embedding substance (for
example O.C.T. compound), and frozen in dry ice/acetone.
Cut sections, mount them on silane-coated glass slides.
If this fixative is used, there are no significant differences of
sensitivity between paraffin-embedded sections and that of frozen sections.
Hybridization (paraffin-embedded sections)
1. Deparaffinize sections in xylene-ethanol series.
2. Heat the sections in lOmM citrate buffer (pH 6.0) by microwave (IS mins after boiling).
3. Wash the sections in PBS.
4. Treat the sections with proteinase K (20)lg/ml in PBS) for 30
mins at 37°C.
5. Wash the sections in PBS, 3x.
6. Fix the sections in 4% PFA in PBS for 5 mins.
7. Immerse the sections in glycine (IOmg/ml) for 15 mins, 2x.
8. Immerse the sections in 2x SSC/50% deionized formamide,
30mins
The procedure step 6-8 can be replaced by dehydration by
ethanol and dry-up. The heating procedure (step 2) is used to
enhance the sensitivity (mildly effective).
For frozen sections, first cut frozen sections, mount them
onto coated-glass slides, and air-dry them for Ihr. When fresh
frozen sections are used, fix tissue sections with 4% PF A for 30
mins. Immerse sections in PBS to remove OCT compound. The
hybridization procedures are the same as those of paraffin-embedded sections except that the condition of proteinase K treatment is mild; i.e., 2)lg/ml for 10 mins at 37°C.
Prehybridization (RNasefree condition)