192
MASANOBU MIYAZAKI, YOSHIYUKI OZONO, TAKASHI HARADA AND SHIGERU KOHNO
Immunological detection with ALP-conjugated anti-DIG antibody after
hybridization followed by washing
1. Sections are reacted with Buffer 2 for 30 mins at room temperature.
2. After draining Buffer 2, sections are incubated with 5,000
times diluted alkali-phosphatase conjugated anti-DIG antibody by Buffer 2 for 60 mins.
3. Washing the sections by Buffer 1.
4. Treat the sections with Buffer 3 for 5 mins.
5. Sections are reacted with color substrate solution. Examine
the signal intensity using a low power magnification, and
stop the color development by washing with Buffer 4.
6. Wash the sections under running water for a short period of
time then dry. Mount in Glycergel (Dako C0563).
Evaluation of specificity of the signal
1. Competitive study: A large amount of unlabeled oligonucleotide (50 - 200 times) is added to the standard hybridization
mixture. Binding of DIG-labeled oligonucleotide to the target
mRNA is inhibited and the signal becomes weak or disappears.
2. Sense probe study: Sense probe, which has a complementary
sequence to anti-sense probe, is used instead of anti-sense
probe. The signal is not detected.
3. RNase treatment: The section is treated with RNase before
hybridization. The hybridization signal is markedly reduced,
indicating that the signal is mRNA-related.
Note: The sequence of probe should be specific to the target
mRNA. Any similarity between the sequence of the probe and
that of any molecules should be checked by computer search
of the GeneBank.
Note: When double strand DNA is used as a probe, it could be
labeled with DIG DNA labeling kit (Boehringer) according the
MASANOBU MIYAZAKI, YOSHIYUKI OZONO, TAKASHI HARADA AND SHIGERU KOHNO
Immunological detection with ALP-conjugated anti-DIG antibody after
hybridization followed by washing
1. Sections are reacted with Buffer 2 for 30 mins at room temperature.
2. After draining Buffer 2, sections are incubated with 5,000
times diluted alkali-phosphatase conjugated anti-DIG antibody by Buffer 2 for 60 mins.
3. Washing the sections by Buffer 1.
4. Treat the sections with Buffer 3 for 5 mins.
5. Sections are reacted with color substrate solution. Examine
the signal intensity using a low power magnification, and
stop the color development by washing with Buffer 4.
6. Wash the sections under running water for a short period of
time then dry. Mount in Glycergel (Dako C0563).
Evaluation of specificity of the signal
1. Competitive study: A large amount of unlabeled oligonucleotide (50 - 200 times) is added to the standard hybridization
mixture. Binding of DIG-labeled oligonucleotide to the target
mRNA is inhibited and the signal becomes weak or disappears.
2. Sense probe study: Sense probe, which has a complementary
sequence to anti-sense probe, is used instead of anti-sense
probe. The signal is not detected.
3. RNase treatment: The section is treated with RNase before
hybridization. The hybridization signal is markedly reduced,
indicating that the signal is mRNA-related.
Note: The sequence of probe should be specific to the target
mRNA. Any similarity between the sequence of the probe and
that of any molecules should be checked by computer search
of the GeneBank.
Note: When double strand DNA is used as a probe, it could be
labeled with DIG DNA labeling kit (Boehringer) according the