56
TAKEHlKO KOJl
Fixation
Protease
treatment
cleotides, which are conserved beyond species (ie, 100% homology of the sequence was found in mouse, rat and human), was
selected and the oligo-DNA complementary to the segment of
28S rRNA with auxiliary ATT repeats at the 3'- and 5'- ends
was synthesized (Yoshii et al. 1995), as follows; 5'-TTATTATGCTACTACCACCAAGATCTGCACCT GCGGCGGC ATTATTATT-3'. Here, ATT repeats were added to be haptenized
with T -T dimer formed by UV irradiation.
With this probe, 28S rRNA was clearly localized in the cytoplasmic area as well as in nucleoli in various cells and tissue sections and we believe that 28S rRNA staining by in situ hybridization is a convenient parameter for evaluation oflevels ofhybridizable RNAs in tissue sections. Also, we confirmed the effectiveness of 28S rRNA staining as reference for optimization of the
pretreatment conditions including protease digestion.
Essential steps in the pretreatment
To immobilize mRNA and to preserve the cell and tissue morphology, fixation is an essential step. As a fixative for in situ hybridization, both protein- precipitating fixatives such as ethanol!
acetic acid (3 : 1) and protein cross-linking fixatives such as
formaldehyde and glutaraldehyde have been used (Koji and Nakane 1990). It should be noted that although stronger fixation
(e.g., a longer exposure of specimens to paraformaldehyde or
fixation with glutaraldehyde) results in higher levels of the retention of cellular RNA, it is known to decrease the penetrability of
probe nucleic acids into cells or tissue sections (Moench et al.
1985; Lawrence and Singer 1985). In our hands, the most favorable fixation is obtained with 4% paraformaldehyde in PBS (pH
7.4) for in situ hybridization of specific RNA. However, we do not
care so much even if we encounter specimens processed under
suboptimal conditions, because we are routinely assessing the
retention of hybridizable RNA, as described above.
In fixed materials, mRNA is supposed to be embedded in cellular
matrix proteins and prevented from forming hybrids with probe
nucleic acids. Consequently, unmasking of mRNAs by removing
proteins from the specimens is often required for full implementation of in situ hybridization. To accomplish this task, the most
TAKEHlKO KOJl
Fixation
Protease
treatment
cleotides, which are conserved beyond species (ie, 100% homology of the sequence was found in mouse, rat and human), was
selected and the oligo-DNA complementary to the segment of
28S rRNA with auxiliary ATT repeats at the 3'- and 5'- ends
was synthesized (Yoshii et al. 1995), as follows; 5'-TTATTATGCTACTACCACCAAGATCTGCACCT GCGGCGGC ATTATTATT-3'. Here, ATT repeats were added to be haptenized
with T -T dimer formed by UV irradiation.
With this probe, 28S rRNA was clearly localized in the cytoplasmic area as well as in nucleoli in various cells and tissue sections and we believe that 28S rRNA staining by in situ hybridization is a convenient parameter for evaluation oflevels ofhybridizable RNAs in tissue sections. Also, we confirmed the effectiveness of 28S rRNA staining as reference for optimization of the
pretreatment conditions including protease digestion.
Essential steps in the pretreatment
To immobilize mRNA and to preserve the cell and tissue morphology, fixation is an essential step. As a fixative for in situ hybridization, both protein- precipitating fixatives such as ethanol!
acetic acid (3 : 1) and protein cross-linking fixatives such as
formaldehyde and glutaraldehyde have been used (Koji and Nakane 1990). It should be noted that although stronger fixation
(e.g., a longer exposure of specimens to paraformaldehyde or
fixation with glutaraldehyde) results in higher levels of the retention of cellular RNA, it is known to decrease the penetrability of
probe nucleic acids into cells or tissue sections (Moench et al.
1985; Lawrence and Singer 1985). In our hands, the most favorable fixation is obtained with 4% paraformaldehyde in PBS (pH
7.4) for in situ hybridization of specific RNA. However, we do not
care so much even if we encounter specimens processed under
suboptimal conditions, because we are routinely assessing the
retention of hybridizable RNA, as described above.
In fixed materials, mRNA is supposed to be embedded in cellular
matrix proteins and prevented from forming hybrids with probe
nucleic acids. Consequently, unmasking of mRNAs by removing
proteins from the specimens is often required for full implementation of in situ hybridization. To accomplish this task, the most