18 Electron Microscopic in Situ Hybridization and its Combination
251
When the frozen sections of A431 tumor were fIxed with 4% paraformaldehyde and reacted with the 91 bp probe, the staining of
perinuclear area as well as nuclei in a speckled pattern (Fig. 1)
was observed and the staining intensity was increased depending
upon the concentrations of the probe and reached a plateau level
at 0.5-1 g/ml. Moreover, the nuclear staining with the probe was
dependent upon a salt concentration and the signal/noise ratio
was a maximum of 150 mM NaCl. The staining with the 91 bp
probe was abolished by the presence of an excess amount of unlabeled 91 bp DNA or unlabeled ETF responsive element DNA
alone, indicating that the nuclear and perinuclear staining with
the 91 bp probe reflects the localization of ETF [12].
A histochemical method for the localization of specifIc DNA- Conclusion
binding proteins by using haptenized synthetic ds-oligo DNA
with specifIc base sequences is introduced. Lists of RE and
RE-binding proteins, including specifIc DNA-binding proteins
that may be involved in the regulation of translation and/or
mRNA stabilization, are rapidly growing. However, antibodies
are not available for most of these newly identifIed binding proteins. Thus, southwestern histochemistry can be a novel tool to
analyze cellular expression of gene-specifIc transcription regulatory factors.
References
1. Lin, c., R., Chen, W. S., Kruiger, W., Stolarsky, L. S., Wdber, W., Evans,
R. M., Verma, I, M., Gill, G. N. and Rosenfeld, M. G.: Expression cloning
of human EGF receptor complementary DNA: gene amplification and
three related messenger RNA products in A431 cells. Science 224:843848, 1984
2. Merlino, G. T., Xu, y, -h., Ishii, S., Clark, A. J. L., Semba, K., Toyoshima,
K., Yamamoto, T. and Pastan, 1.: Amplification and enhanced expression of the epidermql growth factor recptor gene in A431 human carcinoma cells. Science. 224:417-419, 1984.
3. Komuta, K., Koji, T., Izumi, S., Matsumoto, T., Kohara, N., Motojima, K.,
Kanematsu, T. and Nakane, P., K.: Expression of epidermal growth factor receptor messenger RNA in human colorectal carcinomas assessed
by nonradioactive in situ hybridization. Eur. J. Surg. One., 21:269-275,
1995.
4. King, c., R., Kraus, M. H., Willoams, L, T., Merlinl, G. T., Pastan, I, H.
and Anderson, S. A.: Human tumor cell lines with EGF receptor gene
251
When the frozen sections of A431 tumor were fIxed with 4% paraformaldehyde and reacted with the 91 bp probe, the staining of
perinuclear area as well as nuclei in a speckled pattern (Fig. 1)
was observed and the staining intensity was increased depending
upon the concentrations of the probe and reached a plateau level
at 0.5-1 g/ml. Moreover, the nuclear staining with the probe was
dependent upon a salt concentration and the signal/noise ratio
was a maximum of 150 mM NaCl. The staining with the 91 bp
probe was abolished by the presence of an excess amount of unlabeled 91 bp DNA or unlabeled ETF responsive element DNA
alone, indicating that the nuclear and perinuclear staining with
the 91 bp probe reflects the localization of ETF [12].
A histochemical method for the localization of specifIc DNA- Conclusion
binding proteins by using haptenized synthetic ds-oligo DNA
with specifIc base sequences is introduced. Lists of RE and
RE-binding proteins, including specifIc DNA-binding proteins
that may be involved in the regulation of translation and/or
mRNA stabilization, are rapidly growing. However, antibodies
are not available for most of these newly identifIed binding proteins. Thus, southwestern histochemistry can be a novel tool to
analyze cellular expression of gene-specifIc transcription regulatory factors.
References
1. Lin, c., R., Chen, W. S., Kruiger, W., Stolarsky, L. S., Wdber, W., Evans,
R. M., Verma, I, M., Gill, G. N. and Rosenfeld, M. G.: Expression cloning
of human EGF receptor complementary DNA: gene amplification and
three related messenger RNA products in A431 cells. Science 224:843848, 1984
2. Merlino, G. T., Xu, y, -h., Ishii, S., Clark, A. J. L., Semba, K., Toyoshima,
K., Yamamoto, T. and Pastan, 1.: Amplification and enhanced expression of the epidermql growth factor recptor gene in A431 human carcinoma cells. Science. 224:417-419, 1984.
3. Komuta, K., Koji, T., Izumi, S., Matsumoto, T., Kohara, N., Motojima, K.,
Kanematsu, T. and Nakane, P., K.: Expression of epidermal growth factor receptor messenger RNA in human colorectal carcinomas assessed
by nonradioactive in situ hybridization. Eur. J. Surg. One., 21:269-275,
1995.
4. King, c., R., Kraus, M. H., Willoams, L, T., Merlinl, G. T., Pastan, I, H.
and Anderson, S. A.: Human tumor cell lines with EGF receptor gene