13 In Situ Hybridization for RNA: Nonradioactive Probe: Oligo-DNA Probe
189
Note: It does not matter even if the duration of the above step is
longer than 30 mins.
Note: Prehybridization may not be necessary in other protocols.
However, during the preparation for hybridization, it is probably
better to put the sections into a prehybridization buffer than to
leave them in PBS.
9. Boiling the probe in the hybridization buffer: After adjustment of the probe concentration by the prehybridization
buffer, put the solution in the 1.5 ml of eppen-tube and
boil it for 5 mins. Put the tube into ice and water immediately
and leave it for at least 10 mins.
Note: Immediate cooling es essential.
10. Hybridization: React the sections in the hybridization buffer
at 37°C overnight.
Note: Reaction temperature varies depending on the Tm value
(the length of the oligonucleotide and the degree of GC content).
Note: Seal the box tightly. Otherwise, the sections will dry.
Washing and detection of the probe (2nd day)
1. Drain the hybridization buffer on the glass and put it intowashing solution I(WSl). Wash the sections for 30 mins. Repeat the last step again.
2. Wash the sections twice in WS2 for 30 mins each.
Note: Determination of the washing condition is important for
obtaining a good signal. If the washing condition is not tight (low
stringency), the degree of background is higher. Conversely, if it
is too tight (high stringency), the specific signal also becomes
weaker with a low background level.
Note: From this step on, it is not necessary to be careful about
contamination of RNase.
189
Note: It does not matter even if the duration of the above step is
longer than 30 mins.
Note: Prehybridization may not be necessary in other protocols.
However, during the preparation for hybridization, it is probably
better to put the sections into a prehybridization buffer than to
leave them in PBS.
9. Boiling the probe in the hybridization buffer: After adjustment of the probe concentration by the prehybridization
buffer, put the solution in the 1.5 ml of eppen-tube and
boil it for 5 mins. Put the tube into ice and water immediately
and leave it for at least 10 mins.
Note: Immediate cooling es essential.
10. Hybridization: React the sections in the hybridization buffer
at 37°C overnight.
Note: Reaction temperature varies depending on the Tm value
(the length of the oligonucleotide and the degree of GC content).
Note: Seal the box tightly. Otherwise, the sections will dry.
Washing and detection of the probe (2nd day)
1. Drain the hybridization buffer on the glass and put it intowashing solution I(WSl). Wash the sections for 30 mins. Repeat the last step again.
2. Wash the sections twice in WS2 for 30 mins each.
Note: Determination of the washing condition is important for
obtaining a good signal. If the washing condition is not tight (low
stringency), the degree of background is higher. Conversely, if it
is too tight (high stringency), the specific signal also becomes
weaker with a low background level.
Note: From this step on, it is not necessary to be careful about
contamination of RNase.