186
MASANOBU MIYAZAKI, YOSHIYUKI OZONO, TAKASHI HARADA AND SHIGERU KOHNO
- Hybridization buffer:
Final probe concentration is 0.5 - 2.0 ng//ll in the prehybridization buffer.
- Washing solution (WS):
- WS1;2x SSC with 0.075% of BRIJ
- WS2;0.5x SSC with 0.075% of BRIJ
- Preincubation buffer in histochemistry:
- 20% normal swine serum
- 5% BSA
- 5% FCS in PBS
- Dilution buffer of second and third antibodies:
- 4% normal swine serum
- 1% BSA
- 1% FCS in PBS
Procedure
Tissue preparation
The biopsy specimens are embedded in OCT compound and
stored -70°C until use. Fresh frozen tissue is cut into 4 /lm thick
sections and adhered onto APS-coated slide glasses. Dry the
slides well by using cold air of a dryer for at least 30 mins. Sections can be stored in a tight sealed box at -70°C.
Note: The sections can be generally stored for 6 months (Signal
can be obtained).
Note: Tissue samples can be embedded in wax. Fixation before
embedding is important to obtain a good signal. Strong fixation
results in detailed morphology, whereas a low signal may be due
to reduced accessibility of the probe to the target mRNA. We
recommend the use of 4% paraformaldehyde for fixation.
Note: Assessment of remaining RNA in tissue: When a negative
signal is obtained in in situ hybridization, it is difficult to determine whether this represents a lack of the specific mRNA, a technical error or whether mRNA is destroyed in the tissue. Methyl
green-pyronin Y staining is easy to perform and useful to examine the retained RNA (Schulte et al. 1992). Although the pink
color is stained by methyl green-pyronin-Y, indicating preserva-
MASANOBU MIYAZAKI, YOSHIYUKI OZONO, TAKASHI HARADA AND SHIGERU KOHNO
- Hybridization buffer:
Final probe concentration is 0.5 - 2.0 ng//ll in the prehybridization buffer.
- Washing solution (WS):
- WS1;2x SSC with 0.075% of BRIJ
- WS2;0.5x SSC with 0.075% of BRIJ
- Preincubation buffer in histochemistry:
- 20% normal swine serum
- 5% BSA
- 5% FCS in PBS
- Dilution buffer of second and third antibodies:
- 4% normal swine serum
- 1% BSA
- 1% FCS in PBS
Procedure
Tissue preparation
The biopsy specimens are embedded in OCT compound and
stored -70°C until use. Fresh frozen tissue is cut into 4 /lm thick
sections and adhered onto APS-coated slide glasses. Dry the
slides well by using cold air of a dryer for at least 30 mins. Sections can be stored in a tight sealed box at -70°C.
Note: The sections can be generally stored for 6 months (Signal
can be obtained).
Note: Tissue samples can be embedded in wax. Fixation before
embedding is important to obtain a good signal. Strong fixation
results in detailed morphology, whereas a low signal may be due
to reduced accessibility of the probe to the target mRNA. We
recommend the use of 4% paraformaldehyde for fixation.
Note: Assessment of remaining RNA in tissue: When a negative
signal is obtained in in situ hybridization, it is difficult to determine whether this represents a lack of the specific mRNA, a technical error or whether mRNA is destroyed in the tissue. Methyl
green-pyronin Y staining is easy to perform and useful to examine the retained RNA (Schulte et al. 1992). Although the pink
color is stained by methyl green-pyronin-Y, indicating preserva-