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MASANOBU MIYAZAKI, YOSHIYUKI OZONO, TAKASHI HARADA AND SHIGERU KOHNO
Hybridization
1. Take out the tightly sealed box, where the sections were
stored, and warm it to 37°C for 60 mins.
2. Rehydration: soak the sections in PBS for 3 mins.
3. Fixation: fix the sections with 4% PFA in PBS for 15 mins at
room temperature.
4. Washing: Wash the sections three times with PBS for 3 mins
each time.
5. Deproteinization.
Note: This step increases the accessibility of the probe to the target mRNA in the tissue. The optimal treatment of the section
with HCl and protease varies from one tissue to another and depends on the type of sample and condition of fixation. The optimal condition should be determined by each researcher. No
requirement of this step is mentioned in some protocols
when the length of oligonucleotide is less than 100 bases, however, in our experience, nuclei are often stained without deproteinization as false positive. React the sections with 0.2N HCl diluted with distilled water for 15 mins at room temperature. Wash
sections in PBS three times. Treat the sections with 0.5 mg/ml of
proteinase K diluted by PBS for 10 mins.
Note: Prewarm this diluted proteinase K solution for at least 30
mins before use. Otherwise, the reaction may not be enough
when the whole solution is not warmed at 37°C.
6. Wash the sections in PBS three times.
7. Post fixation: Fix the sections in 4% PFA in PBS again for 10
mins at room temperature. Wash them in PBS three times.
Note: Sometimes after washing, treat the sections with 2 mg/ml
of glycine in PBS for 15 mins twice in order to neutralize aldehyde completely. In our protocol, treatment with glycine is notperformed.
8. Prehybridization: Soak the sections in the prehybridization
buffer for at least 30 mins.
MASANOBU MIYAZAKI, YOSHIYUKI OZONO, TAKASHI HARADA AND SHIGERU KOHNO
Hybridization
1. Take out the tightly sealed box, where the sections were
stored, and warm it to 37°C for 60 mins.
2. Rehydration: soak the sections in PBS for 3 mins.
3. Fixation: fix the sections with 4% PFA in PBS for 15 mins at
room temperature.
4. Washing: Wash the sections three times with PBS for 3 mins
each time.
5. Deproteinization.
Note: This step increases the accessibility of the probe to the target mRNA in the tissue. The optimal treatment of the section
with HCl and protease varies from one tissue to another and depends on the type of sample and condition of fixation. The optimal condition should be determined by each researcher. No
requirement of this step is mentioned in some protocols
when the length of oligonucleotide is less than 100 bases, however, in our experience, nuclei are often stained without deproteinization as false positive. React the sections with 0.2N HCl diluted with distilled water for 15 mins at room temperature. Wash
sections in PBS three times. Treat the sections with 0.5 mg/ml of
proteinase K diluted by PBS for 10 mins.
Note: Prewarm this diluted proteinase K solution for at least 30
mins before use. Otherwise, the reaction may not be enough
when the whole solution is not warmed at 37°C.
6. Wash the sections in PBS three times.
7. Post fixation: Fix the sections in 4% PFA in PBS again for 10
mins at room temperature. Wash them in PBS three times.
Note: Sometimes after washing, treat the sections with 2 mg/ml
of glycine in PBS for 15 mins twice in order to neutralize aldehyde completely. In our protocol, treatment with glycine is notperformed.
8. Prehybridization: Soak the sections in the prehybridization
buffer for at least 30 mins.