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HARUO OHTANI
4. Add 10).11 of 3M sodium acetate, 5).11 of 20mg/ml and E. coli
tRNA and 350).11 of chilled ethanol. Stay at -70°C for 2hrs
or at -20°C overnight. Microfuge at 14,000rpm for 15
mins. Rinse the precipitate with 70-80% ethanol. Dry up. Dissolve the precipitate with 50).11 of nuclease free water (or TE
buffer) containing 20U RNase inhibitor. Store at -20 or -70°e.
Quantification of the cRNAs by immunohistochemical staining
1. Dilute the solution at 1:100, 1:200, 1:400, 1:800, and 1:1600. The
dilution buffer is made by adding 5).11 of 1 Omg/ml E. coli tRN A
into 1000).11 of TE buffer.
2. Mark them to the filter membrane (1).11) and crosslink them by
ultraviolet.
3. Stain the filter with anti-digoxigenin antibody, and develop
the color reagents by alkaline phosphatase substrate.
4. Compare each reaction with that of control labeled RNA.
Fixation
Both paraffin -embedded sections and frozen sections can be
used.
The most frequently used fixative is 4% paraformaldehyde
solution (PF A)/phosphate buffer. The fixation time is 2hrs to
overnight at 4°e.
Our current fixatives are 4%PF A + 0.5% glutaraldehyde/PBS
(0.075M, pH 7.2) containing 1mM MgCh and 1mM CaC12• This
fixation is quite useful because of higher sensitivity and more
consistent results 1 ,2). Immerse sliced tissues in this fixative overnight at 4 0e. The thickness of tissue should be 2mm. If thick
(>2mm) sections were fixed, uneven fixation results will be
gained. If possible, perfusion fixation is more preferable. This
fixative is useful for the fixation of bone tissue followed by
EDTA decalcification 3 ).
After the completion of fixation, the specimens are either dehydrated by ethanol to be embedded in paraffin or frozen. For
frozen sections, the specimens are washed in 20-30% sucrose/
HARUO OHTANI
4. Add 10).11 of 3M sodium acetate, 5).11 of 20mg/ml and E. coli
tRNA and 350).11 of chilled ethanol. Stay at -70°C for 2hrs
or at -20°C overnight. Microfuge at 14,000rpm for 15
mins. Rinse the precipitate with 70-80% ethanol. Dry up. Dissolve the precipitate with 50).11 of nuclease free water (or TE
buffer) containing 20U RNase inhibitor. Store at -20 or -70°e.
Quantification of the cRNAs by immunohistochemical staining
1. Dilute the solution at 1:100, 1:200, 1:400, 1:800, and 1:1600. The
dilution buffer is made by adding 5).11 of 1 Omg/ml E. coli tRN A
into 1000).11 of TE buffer.
2. Mark them to the filter membrane (1).11) and crosslink them by
ultraviolet.
3. Stain the filter with anti-digoxigenin antibody, and develop
the color reagents by alkaline phosphatase substrate.
4. Compare each reaction with that of control labeled RNA.
Fixation
Both paraffin -embedded sections and frozen sections can be
used.
The most frequently used fixative is 4% paraformaldehyde
solution (PF A)/phosphate buffer. The fixation time is 2hrs to
overnight at 4°e.
Our current fixatives are 4%PF A + 0.5% glutaraldehyde/PBS
(0.075M, pH 7.2) containing 1mM MgCh and 1mM CaC12• This
fixation is quite useful because of higher sensitivity and more
consistent results 1 ,2). Immerse sliced tissues in this fixative overnight at 4 0e. The thickness of tissue should be 2mm. If thick
(>2mm) sections were fixed, uneven fixation results will be
gained. If possible, perfusion fixation is more preferable. This
fixative is useful for the fixation of bone tissue followed by
EDTA decalcification 3 ).
After the completion of fixation, the specimens are either dehydrated by ethanol to be embedded in paraffin or frozen. For
frozen sections, the specimens are washed in 20-30% sucrose/