18 Electron Microscopic in Situ Hybridization and its Combination
245
acted haptenized double-stranded DNAs with a section and examined a signal immunochistochemically.
Procedure
Practice of Southwestern histochemistry
The contamination of RNase may be admitted. However, a contamination of DNase should be avoided. Actually, we experiment
in the same room as for an immunohistochemical experiment.
However, a solution is treated by autoclave and sterilized
glasses are used.
We synthesized the set of (+) and (-) oligo-DNAs consisting of
specific responsive element on an automated DNA synthesizer
(Applied Biosystems, Model 391 PCR-Mate) . These synthesized
oligo-DNAs were purified by an OPC column.
(a) Thymine-thymine (T-T) dimers
For haptenization with T -T dimers of the oligo-DNAs, we added
2 and 3 TTA repeats at 5',- and 3',- end of the selected sequences,
respectively. The diluted (20-50 /lsg/ml) oligo-DNA by UV irradiation (5000-10000 J/m2 ) was haptenized. The optimal UV dose
was determined by dot-blot hybridization [6].
(b) Dig-11-dUTP
The synthesized oligo-DNAs were labeled at 3' -end with Dig-lldUTP by terminal deoxynucleotidyl transferase [7]. The haptenization was confirmed by dot-blot hybridization.
Annealing of single-stranded(ss) oligo-DNAs to double stranded
(ds) oligo-DNA. The (+) and (-) strands of each synthesized oligo-DNAs dissolved in 10 mM Tris-HCI buffer(pH7.4) containing
1 mM EDTA were denatured for 10 min at (Tm-25) °C and thereafter gradually cooled to 20°C. Then the re-annealing confirmed
that the oligo-DNAs were separated by PAGE with 4-20 % gradient gel and were stained with the silver staining kit (Daiichi
Pure Chern. Co., Japan).
Room
Synthesis of
oligodeoxynucleotide
(oligo-DNA)
Haptenization
of oligo-DNA
Annealing
Aliquotes of the nuclear extract in 62.5 mM Tris/HCI (pH6.8) Southwestern
containing 2% SDS,15% glycerol, and 0.0025% bromophenol blot analysis
245
acted haptenized double-stranded DNAs with a section and examined a signal immunochistochemically.
Procedure
Practice of Southwestern histochemistry
The contamination of RNase may be admitted. However, a contamination of DNase should be avoided. Actually, we experiment
in the same room as for an immunohistochemical experiment.
However, a solution is treated by autoclave and sterilized
glasses are used.
We synthesized the set of (+) and (-) oligo-DNAs consisting of
specific responsive element on an automated DNA synthesizer
(Applied Biosystems, Model 391 PCR-Mate) . These synthesized
oligo-DNAs were purified by an OPC column.
(a) Thymine-thymine (T-T) dimers
For haptenization with T -T dimers of the oligo-DNAs, we added
2 and 3 TTA repeats at 5',- and 3',- end of the selected sequences,
respectively. The diluted (20-50 /lsg/ml) oligo-DNA by UV irradiation (5000-10000 J/m2 ) was haptenized. The optimal UV dose
was determined by dot-blot hybridization [6].
(b) Dig-11-dUTP
The synthesized oligo-DNAs were labeled at 3' -end with Dig-lldUTP by terminal deoxynucleotidyl transferase [7]. The haptenization was confirmed by dot-blot hybridization.
Annealing of single-stranded(ss) oligo-DNAs to double stranded
(ds) oligo-DNA. The (+) and (-) strands of each synthesized oligo-DNAs dissolved in 10 mM Tris-HCI buffer(pH7.4) containing
1 mM EDTA were denatured for 10 min at (Tm-25) °C and thereafter gradually cooled to 20°C. Then the re-annealing confirmed
that the oligo-DNAs were separated by PAGE with 4-20 % gradient gel and were stained with the silver staining kit (Daiichi
Pure Chern. Co., Japan).
Room
Synthesis of
oligodeoxynucleotide
(oligo-DNA)
Haptenization
of oligo-DNA
Annealing
Aliquotes of the nuclear extract in 62.5 mM Tris/HCI (pH6.8) Southwestern
containing 2% SDS,15% glycerol, and 0.0025% bromophenol blot analysis