60
TAKEHIKO KOJI
body and the third HRP-Iabeled antibody, seemed effective to
some extent (Miyazaki et al. 1996).
Very recently, Kerstens et al. (1995) have reported the use of
biotinylated tyramine as a system of catalyzed reporter deposition in chromosomal in situ hybridization. More recently, De
Haas et al. (1996) have found the effectiveness of the system
with biotinylated tyramine in fluorescence in situ hybridization
for specific RNA sequences in cultured cells. In our trial, we examined the usefulness of catalyzed signal amplification (CSA) or
catalyzed reporter deposition system with biotinylated tyramine
to amplify colorimetric in situ hybridization signals, using part
of a DAKO kit. Our CSA protocol included the use ofbiotinylated
tyramine after the reaction of T -T dimerized DNA with HRP-Iabeled anti-(T-T dimer) antibody, followed by the reaction with
HRP-Iabeled streptavidin. When T-T dimerized A phage DNA
was spotted onto a nitrocellulose filter at 1 pg - 10 ng and
then detected by the direct immunoperoxidase method, the indirect immunoperoxidase method or the CSA method, the CSA
method gave the highest sensitivity with about 100-fold increase
compared to that of the indirect method (Koji et al. 1997). Next,
in the frozen sections of rat brain, which was fixed with 4% paraformaldehyde, 28S rRNA staining by in situ hybridization with
only 10 ng/ml of T -T dimerized oligonucleotide probe complementary to rat 28S rRNA was compared with the direct method,
the HRP-Iabeled avidin-biotin complex method and the CSA
method. Again, intense 28S rRNA signal was obtained only
with the CSA method, as shown in Fig. 3. Therefore, we confirmed the usefulness of the catalyzed deposition system with
biotinylated tyramine in nonradioactive colorimetric in situ hybridization for specific RNA sequences in tissue sections.
Further studies will be necessary to manage the development
of background staining for a wide application.
When a probe labeled with some fluorescent dye is used, one
can expect a direct detection of the signal by a fluorescent microscope or a laser scanning microscopy. Actually, it has turned
out to be very efficient in chromosomal in situ hybridization for
localization of specific sequences of genomic DNA in chromosomes (Ried et al. 1992). Very interestingly, however, a similar
approach has often failed to localize specific mRNA, partly because of high background fluorescence. Therefore, even if a
fluorochrome-labeled probe is used, the signal detection is
TAKEHIKO KOJI
body and the third HRP-Iabeled antibody, seemed effective to
some extent (Miyazaki et al. 1996).
Very recently, Kerstens et al. (1995) have reported the use of
biotinylated tyramine as a system of catalyzed reporter deposition in chromosomal in situ hybridization. More recently, De
Haas et al. (1996) have found the effectiveness of the system
with biotinylated tyramine in fluorescence in situ hybridization
for specific RNA sequences in cultured cells. In our trial, we examined the usefulness of catalyzed signal amplification (CSA) or
catalyzed reporter deposition system with biotinylated tyramine
to amplify colorimetric in situ hybridization signals, using part
of a DAKO kit. Our CSA protocol included the use ofbiotinylated
tyramine after the reaction of T -T dimerized DNA with HRP-Iabeled anti-(T-T dimer) antibody, followed by the reaction with
HRP-Iabeled streptavidin. When T-T dimerized A phage DNA
was spotted onto a nitrocellulose filter at 1 pg - 10 ng and
then detected by the direct immunoperoxidase method, the indirect immunoperoxidase method or the CSA method, the CSA
method gave the highest sensitivity with about 100-fold increase
compared to that of the indirect method (Koji et al. 1997). Next,
in the frozen sections of rat brain, which was fixed with 4% paraformaldehyde, 28S rRNA staining by in situ hybridization with
only 10 ng/ml of T -T dimerized oligonucleotide probe complementary to rat 28S rRNA was compared with the direct method,
the HRP-Iabeled avidin-biotin complex method and the CSA
method. Again, intense 28S rRNA signal was obtained only
with the CSA method, as shown in Fig. 3. Therefore, we confirmed the usefulness of the catalyzed deposition system with
biotinylated tyramine in nonradioactive colorimetric in situ hybridization for specific RNA sequences in tissue sections.
Further studies will be necessary to manage the development
of background staining for a wide application.
When a probe labeled with some fluorescent dye is used, one
can expect a direct detection of the signal by a fluorescent microscope or a laser scanning microscopy. Actually, it has turned
out to be very efficient in chromosomal in situ hybridization for
localization of specific sequences of genomic DNA in chromosomes (Ried et al. 1992). Very interestingly, however, a similar
approach has often failed to localize specific mRNA, partly because of high background fluorescence. Therefore, even if a
fluorochrome-labeled probe is used, the signal detection is