108
TAKASHI SAWAI AND MIWA UZUKI
Prehybridization
treatment
Prepare the
hybridization
mixture
1. Cover the sample with predigested pronase* for 10 mins at
room temperature
Note: Dissolve 10 mg pronase (Calbiochem) in 4 ml H20 and
incubate for 4 hrs at 37°C. Divide into 40 aliquots of 100).11. Store
at -20°C. Before use, mix one aliquot (0.25 mgt 100).11 H20) with
900 ).11 of 50mM Tris-HC1, pH 7.6, containing 5mM EDTA.
2. Wash in PBS containing 2 mg/ml glycine (30 sec x2) and in
PBS (30 secs x2) (Stop the enzymic reaction and neutralize
excess aldehyde residue)
3. Fix in 4% paraformaldehyde in PBS for 20 mins
4. Wash in PBS containing 2 mg/ml glycine (5 mins x2)
5. Immerse in 0.1 M triethanolamine buffer (pH 8.0) for 5 mins
6. Immerse in a freshly prepared mixture* of 0.25% acetic anhydride in the triethanolamine buffer for 10 mins with occasional shaking.
Note: To prepare the mixture, acetic anhydride should be added
in a few minutes because half-life of this mixture is 1 min. Mix
within few secs before use
7. Wash in 2 x SSC (5 mins x2)
8. Dehydrate in 70% ethanol (5 mins x2) and in 95% ethanol (5
mins)
9. Dry in air (section side down)
For negative control, after washing three times with PBS, slides
are reacted with RNase solution (2 mg/m1300mM Tris-NaCl, pH
7.5) ,wash in PBS again, then dehydrate in 70% ethanol (5 mins
x2) and in 95% ethanol,S mins and dry in air.
Probe
0.1 flg
ssDNA
8 flg
Formamide
50 fll
TNEB + DS >(50 fll
Yeast tRNA (55mg/ml)
1 fll