58
TAKEHIKO KOJI
mRNA is preincubated or added to the hybridization mixture and the signal is abolished or markedly decreased, the
signal is considered to be specific. In the blocking control, we
should show that non-labeled unrelated nucleic acid cannot
block the successive hybridization with the proper probe. In
the competition experiment, it is also essential to show that
no changes occur in the presence of an excess amount of
non-labeled unrelated nucleic acid, neglecting the effect
of simple dilution of the proper probe.
(d) Excessively high stringent washing. When the cells or tissue
sections, which were already hybridized with a specific
probe, are washed at a higher temperature than the expected
Tm value of the hybrids, the signal should be dislodged.
(e) Biological control. No positive signal is found in specimens
which are known to lack the specific target mRNA.
(f) If possible, it should be demonstrated that the target tissue
expresses only one species of mRNA which hybridizes with
the probe by Northern blotting.
I will show an example of a set of control experiments in Fig. 2,
where estrogen receptor mRNA was localized in situ with Diglabeled antisense oligo-DNA probe in the fresh frozen sections of
monkey uterus (Koji and Brenner 1993).
New approaches to increase the sensitivity of in situ hybridization
In our routine protocol, the sites of HRP are visualized with hydrogen peroxide and 3,3' -diaminobenzidine (DAB) in the presence of nickel and cobalt ions (Adams 1981), which increases
the sensitivity more than ten-fold as compared to that of hydrogen peroxide and DAB alone. Under our conditions detailed previously (Koji and Nakane 1990), 1 - 10 pg of DNA Is pot was usually detected in dot -blot hybridization. However, it was sometimes
felt that the sensitivity was still not enough. We attempted various signal enhancing protocols including silver enhancement
and colloidal gold-silver enhancement, but such trials were
not successful in our hands. Triple layered method, where the
reaction with the first antibody is followed by the second anti-
TAKEHIKO KOJI
mRNA is preincubated or added to the hybridization mixture and the signal is abolished or markedly decreased, the
signal is considered to be specific. In the blocking control, we
should show that non-labeled unrelated nucleic acid cannot
block the successive hybridization with the proper probe. In
the competition experiment, it is also essential to show that
no changes occur in the presence of an excess amount of
non-labeled unrelated nucleic acid, neglecting the effect
of simple dilution of the proper probe.
(d) Excessively high stringent washing. When the cells or tissue
sections, which were already hybridized with a specific
probe, are washed at a higher temperature than the expected
Tm value of the hybrids, the signal should be dislodged.
(e) Biological control. No positive signal is found in specimens
which are known to lack the specific target mRNA.
(f) If possible, it should be demonstrated that the target tissue
expresses only one species of mRNA which hybridizes with
the probe by Northern blotting.
I will show an example of a set of control experiments in Fig. 2,
where estrogen receptor mRNA was localized in situ with Diglabeled antisense oligo-DNA probe in the fresh frozen sections of
monkey uterus (Koji and Brenner 1993).
New approaches to increase the sensitivity of in situ hybridization
In our routine protocol, the sites of HRP are visualized with hydrogen peroxide and 3,3' -diaminobenzidine (DAB) in the presence of nickel and cobalt ions (Adams 1981), which increases
the sensitivity more than ten-fold as compared to that of hydrogen peroxide and DAB alone. Under our conditions detailed previously (Koji and Nakane 1990), 1 - 10 pg of DNA Is pot was usually detected in dot -blot hybridization. However, it was sometimes
felt that the sensitivity was still not enough. We attempted various signal enhancing protocols including silver enhancement
and colloidal gold-silver enhancement, but such trials were
not successful in our hands. Triple layered method, where the
reaction with the first antibody is followed by the second anti-