190
MASANOBU MIYAZAKI, YOSHIYUKI OZONO, TAKASHI HARADA AND SHIGERU KOHNO
Visualization of the probe bound to specific mRNA on the tissue
Using immunohistochemistry, the probe bound to the specific
mRNA in the tissue can be visualized using a light microscope.
In our protocol, three-layer immunoperoxidase method with
anti-DIG antibody and HRP-conjugated antibodies is performed
to amplify the signal.
Note: The nonradioactive in situ hybridization method is generally considered to be less sensitive than the radioactive one.
Our three-layer immunoperoxidase system is essential for the
detection of hybridized probe. It remarkably increases the sensitivity of in situ hybridization using DIG-labeled oligonucleotide probe. Furthermore, in our original method, a four-layer
immunoperoxidase system was used for the detection of the
probe to enhance the sensitivity (Miyazaki et al. 1994).
1. Preincubation: After washing, put the preincubation buffer
on the slide and leave it for at least 60 mins at room temperature.
Note: Do not dry the sections, otherwise non-specific signal develops.
2. Reaction with the anti-DIG antibody: Drain the preincubation
buffer and react the sections with the monoclonal anti-DIG
antibody diluted by the dilution buffer (final concentration;
1 /lg/ml) for at least 60 mins at room temperature. Wash by
PBS for 10 mins four times.
Note: HRP-conjugated sheep anti-DIG (Fab fragment) is also
available from Boehringer, however, in immunohistochemistry
this antibody is much less sensitive.
3. Methanol blocking: In order to block endogenous peroxidase,
the sections are reacted with methanol containing 0.3% H20 2
for 15 mins at room temperature.
Note: In this step, sections must be shielded. After methanol
blocking, wash the sections three t~mes in PBS, each for 3 mins.
4. Reaction with second and third antibodies: Sections are reacted with one hundred times diluted HRP-conjugated rabbit
anti-mouse immunoglobulin antibody for 30 mins at room
temperature. After washing three times with PBS containing
MASANOBU MIYAZAKI, YOSHIYUKI OZONO, TAKASHI HARADA AND SHIGERU KOHNO
Visualization of the probe bound to specific mRNA on the tissue
Using immunohistochemistry, the probe bound to the specific
mRNA in the tissue can be visualized using a light microscope.
In our protocol, three-layer immunoperoxidase method with
anti-DIG antibody and HRP-conjugated antibodies is performed
to amplify the signal.
Note: The nonradioactive in situ hybridization method is generally considered to be less sensitive than the radioactive one.
Our three-layer immunoperoxidase system is essential for the
detection of hybridized probe. It remarkably increases the sensitivity of in situ hybridization using DIG-labeled oligonucleotide probe. Furthermore, in our original method, a four-layer
immunoperoxidase system was used for the detection of the
probe to enhance the sensitivity (Miyazaki et al. 1994).
1. Preincubation: After washing, put the preincubation buffer
on the slide and leave it for at least 60 mins at room temperature.
Note: Do not dry the sections, otherwise non-specific signal develops.
2. Reaction with the anti-DIG antibody: Drain the preincubation
buffer and react the sections with the monoclonal anti-DIG
antibody diluted by the dilution buffer (final concentration;
1 /lg/ml) for at least 60 mins at room temperature. Wash by
PBS for 10 mins four times.
Note: HRP-conjugated sheep anti-DIG (Fab fragment) is also
available from Boehringer, however, in immunohistochemistry
this antibody is much less sensitive.
3. Methanol blocking: In order to block endogenous peroxidase,
the sections are reacted with methanol containing 0.3% H20 2
for 15 mins at room temperature.
Note: In this step, sections must be shielded. After methanol
blocking, wash the sections three t~mes in PBS, each for 3 mins.
4. Reaction with second and third antibodies: Sections are reacted with one hundred times diluted HRP-conjugated rabbit
anti-mouse immunoglobulin antibody for 30 mins at room
temperature. After washing three times with PBS containing