11 In Situ Hybridization for RNA: Nonradioactive Probe: Oligo-DNA Probe
159
- the constituents of blocking solution
Blocking solution
5% BSA in PBS
500 Ilg/ml normal mouse IgG
100 Ilg/ml yeast tRNA
100 Ilg/ml salmon testicular DNA
0.3 M NaCl
Note. Salmon sperm DNA and yeast tRNA are added to hybridization
medium, blocking solution, and antibody solution as competitive inhibitors of DNase and RNase.
- HRP-labeled anti-(T-T dimer) monoclonal antibody (Kyowa
Medex)
- Triton X-lOO (Sigma)
- Proteinase K (Sigma)
- PF A/PBS (PF A; MERIK)
- 2mg/ml glycine/PBS (glycine; Wako)
- deionized formamide (formamide; Nakarai)
Procedure
T-T dimerization of oligo-DNA probes
Oligo-DNA probes are required about 45-base sequence complementaryto the target mRNAs. To obtain the maximum signal by
in situ hybridization, it should be verified whether the probes are
optimally labeled. Too little labeling results in a low signal,
whereas, excessive labeling will reduce the efficiency of hybrid ization. Antisense and sense sequences are used for synthetic
oligo-DNA probes but for haptenization of the oligo-DNAs
with T -T dimers, two and three TTA repeats sequences are added
at the 5' - and 3' -ends of the sequences, respectively at the time of
synthesis. Recently, it has been possible to synthesize the oligoDNAs with an automatic DNA synthesizer.
for accessories
159
- the constituents of blocking solution
Blocking solution
5% BSA in PBS
500 Ilg/ml normal mouse IgG
100 Ilg/ml yeast tRNA
100 Ilg/ml salmon testicular DNA
0.3 M NaCl
Note. Salmon sperm DNA and yeast tRNA are added to hybridization
medium, blocking solution, and antibody solution as competitive inhibitors of DNase and RNase.
- HRP-labeled anti-(T-T dimer) monoclonal antibody (Kyowa
Medex)
- Triton X-lOO (Sigma)
- Proteinase K (Sigma)
- PF A/PBS (PF A; MERIK)
- 2mg/ml glycine/PBS (glycine; Wako)
- deionized formamide (formamide; Nakarai)
Procedure
T-T dimerization of oligo-DNA probes
Oligo-DNA probes are required about 45-base sequence complementaryto the target mRNAs. To obtain the maximum signal by
in situ hybridization, it should be verified whether the probes are
optimally labeled. Too little labeling results in a low signal,
whereas, excessive labeling will reduce the efficiency of hybrid ization. Antisense and sense sequences are used for synthetic
oligo-DNA probes but for haptenization of the oligo-DNAs
with T -T dimers, two and three TTA repeats sequences are added
at the 5' - and 3' -ends of the sequences, respectively at the time of
synthesis. Recently, it has been possible to synthesize the oligoDNAs with an automatic DNA synthesizer.
for accessories