176
YUKA IWASAKI AND IHACHI KATO
Synthesis and
labeling of
oligo-ONAs
Immunohistochemical
detection of
probes on
nitrocellulose
membranes
The recommended protocol is described for preparation of
the decalcified tissues.
1. Fix in 4% PFA/PBS at 4°C for 12 h and wash with DDW.
2. Decalcify with 10% EDTA at 4°C for 5-6 days and wash with
DDW at 4°C for 12 h.
3. Dehydrate with graded ethanols and xylene, and embed in
paraffin wax.
Oligo-DNA probes require about 45-base sequence complementary to the target mRN As. We chose a sequence according to the
published cDNA base sequence data and synthesized it on a DNA
synthesizer, or obtained it commercially. We also prepared an
oligo-DNA with 34 bases, which is complementary to the part
of 28S rRNA, as a positive control probe, as described in detail
by Y oshii et al. (1995). This control probe is used to assess the
integrity and hybridizability of RNA in tissue sections. In addition, we conducted a computer-assisted search of the oligo-DNA
sequences and no significant homology with any other published
sequence was found.
The oligo-DNAs are labeled by a DIG oligonucleotide tailing
kit according to standard protocols. After separation from the
unreacted compounds by ethanol precipitation, the final precipitates are suspended in TE buffer (10 mM Tris-HCI, pH 7.4, containing 1 mM EDT A) at a final concentration of 25 Ilg/ml.
To confirm the labeling of probe oligo-DNAs, we perform an immunohistochemical detection method.
1. Cut 1 x 4 cm strips of nitrocellulose filters and immerse in
DDW for 30 mins at RT.
2. Replace DDW for 20 x SSC and keep them for 30 mins at RT.
3. Place the filters on a sheet of Parafilm and dry up.
4. Apply 2 III of DNA solution containing digoxigenin-Iabeled
oligo-DNAs onto the filter at various quantities per dot (1 pg,
10 pg, 100 pg, 1 ng, 10 ng).
5. Dry up completely (overnight air-dry is recommended).
6. Bake the filter at 80
D
C for 2 hrs.
7. Irradiate the filter for 1 min.
YUKA IWASAKI AND IHACHI KATO
Synthesis and
labeling of
oligo-ONAs
Immunohistochemical
detection of
probes on
nitrocellulose
membranes
The recommended protocol is described for preparation of
the decalcified tissues.
1. Fix in 4% PFA/PBS at 4°C for 12 h and wash with DDW.
2. Decalcify with 10% EDTA at 4°C for 5-6 days and wash with
DDW at 4°C for 12 h.
3. Dehydrate with graded ethanols and xylene, and embed in
paraffin wax.
Oligo-DNA probes require about 45-base sequence complementary to the target mRN As. We chose a sequence according to the
published cDNA base sequence data and synthesized it on a DNA
synthesizer, or obtained it commercially. We also prepared an
oligo-DNA with 34 bases, which is complementary to the part
of 28S rRNA, as a positive control probe, as described in detail
by Y oshii et al. (1995). This control probe is used to assess the
integrity and hybridizability of RNA in tissue sections. In addition, we conducted a computer-assisted search of the oligo-DNA
sequences and no significant homology with any other published
sequence was found.
The oligo-DNAs are labeled by a DIG oligonucleotide tailing
kit according to standard protocols. After separation from the
unreacted compounds by ethanol precipitation, the final precipitates are suspended in TE buffer (10 mM Tris-HCI, pH 7.4, containing 1 mM EDT A) at a final concentration of 25 Ilg/ml.
To confirm the labeling of probe oligo-DNAs, we perform an immunohistochemical detection method.
1. Cut 1 x 4 cm strips of nitrocellulose filters and immerse in
DDW for 30 mins at RT.
2. Replace DDW for 20 x SSC and keep them for 30 mins at RT.
3. Place the filters on a sheet of Parafilm and dry up.
4. Apply 2 III of DNA solution containing digoxigenin-Iabeled
oligo-DNAs onto the filter at various quantities per dot (1 pg,
10 pg, 100 pg, 1 ng, 10 ng).
5. Dry up completely (overnight air-dry is recommended).
6. Bake the filter at 80
D
C for 2 hrs.
7. Irradiate the filter for 1 min.