11 In Situ Hybridization for RNA: Nonradioactive Probe: Oligo-DNA Probe
163
7. Hybridization. Replace the prehybridization buffer and incubate with hybridization buffer for overnight at 42°e in the
vinyl bag (see Table 2. Hybridization solution).
Table 2. Hybridization solution
Hybridization mediums
Reagents
volume (I-.tl)
lOmM TrislHCI (pH 7.4)
0.6M NaCI
1M TrislHCI (pH 7.4)
2.5
5M NaCI
30
1mM EDTA
200mM EDT A
1.25
40% Deionized formamide
Deionized formamide
100
Ix Denhardt's solution
100x Denhardt's solution
2.5
250 J.lg/ml yeast tRNA
lOmg/ml yeast tRNA
6.25
125 J.lg/ml salmon sperm DNA lOmg/ml salmon
sperm DNA
10% Dextran sulfate
50% Dextran sulfate
1 J.lg/ml T -T dimerized probe 50J.lg/ml T -T dimerized
probe
TE buffer
total (for 1 bag)
Note. The total mixture should be boiled for 7 mins.
3.125
50.0
5.0
49.375
250
Note. It may be desirable to limit Hybridization time to 12 hours.
8. Wash the filter successively as follows.
9. 2x sse 15 mins, twice, RT.
10. Ix sse 15 mins, twice, RT.
11. 0.5 x sse 15 mins, once, RT
12. PBS, 15 mins, once, R T
13. Incubate the filter with a blocking solution consisting of 5%
BSA (98-99% pure), 500 /-lg/ml normal goat IgG, 100 /-lg/ml
yeast tRNA, 100 /-lg/ml salmon sperm DNA, 0.05% NaN3 and
PBS, for 1hr at RT in a vinyl bag.
14. Apply the 1st antibody (mouse anti-T-T IgG: 1:80) dissolved
in PBS consisting 5% BSA, 100 /-lg/ml yeast tRNA, 100 /-lg/ml
Day 3