164
YOSHITAKA HISHIKAWA ET AL.
Recommended protocol
salmon sperm DNA and 0.05% NaN3 for 3 hrs at RT in vinyl
bag.
15. Wash the reacted filter 4 times in PBS for 15 mins each at RT.
16. Apply the 2nd antibody (HRP-goat anti mouse IgG) dissolved in PBS consisting 5% BSA, 100 Ilg/ml yeast tRNA
and 100 Ilg/ml salmon sperm DNA for 1 hr at RT in a vinyl
bag.
17. Repeat step 11.
18. Immerse the filter in the DAB/H20 2/Co/Ni solution for 10
mins at RT (Adams 1981).
19. Wash the filter at least 3 times with DDW for 10 mins each
and allow it to dry.
Localization In Situ of Specific mRNA Using T-T Dimerized Olio-DNA
Probes
Note: The contamination with DNase and RNase should be minimized throughout the in situ hybridization procedure. The
mRNAs are very unstable, so it is necessary to inhibit endogenous RNase activity as much as possible and to remove RNase
contaminsation of the specimens and all equipment. All glassware is baked at 240°C for at least 4 hrs, and the solutions (PBS
and DDW) and disposable tips are autoclaved at 121 °C for 20
mins. The wearing of disposable plastic gloves (not steriled)
is recommended throughout the experiment. Take care when
handling H20 2 and DAB solutions because they are thought
to be carcinogen.
Note: To estimate the adequate amount of mRNA level in paraffin sections, Methyl Green/Pyronin Y staining (Shulte 1992)
should be used before performing in situ hybridization in every
run.
Unless otherwise specified, all steps should be performed at
room temperature.
Pretreatment
1. Cut sections to a thickness of 5 11m and place them on silanecoated glass slides.
YOSHITAKA HISHIKAWA ET AL.
Recommended protocol
salmon sperm DNA and 0.05% NaN3 for 3 hrs at RT in vinyl
bag.
15. Wash the reacted filter 4 times in PBS for 15 mins each at RT.
16. Apply the 2nd antibody (HRP-goat anti mouse IgG) dissolved in PBS consisting 5% BSA, 100 Ilg/ml yeast tRNA
and 100 Ilg/ml salmon sperm DNA for 1 hr at RT in a vinyl
bag.
17. Repeat step 11.
18. Immerse the filter in the DAB/H20 2/Co/Ni solution for 10
mins at RT (Adams 1981).
19. Wash the filter at least 3 times with DDW for 10 mins each
and allow it to dry.
Localization In Situ of Specific mRNA Using T-T Dimerized Olio-DNA
Probes
Note: The contamination with DNase and RNase should be minimized throughout the in situ hybridization procedure. The
mRNAs are very unstable, so it is necessary to inhibit endogenous RNase activity as much as possible and to remove RNase
contaminsation of the specimens and all equipment. All glassware is baked at 240°C for at least 4 hrs, and the solutions (PBS
and DDW) and disposable tips are autoclaved at 121 °C for 20
mins. The wearing of disposable plastic gloves (not steriled)
is recommended throughout the experiment. Take care when
handling H20 2 and DAB solutions because they are thought
to be carcinogen.
Note: To estimate the adequate amount of mRNA level in paraffin sections, Methyl Green/Pyronin Y staining (Shulte 1992)
should be used before performing in situ hybridization in every
run.
Unless otherwise specified, all steps should be performed at
room temperature.
Pretreatment
1. Cut sections to a thickness of 5 11m and place them on silanecoated glass slides.