170
YOSHITAKA HISHIKA '" A ET AL.
• To determine the optimal condition of in situ hybridization,
positive control probe should be used.
T rou bleshooti ng
• With the antisense probe, none or weak staining is found.
- Inadequate amount of mRNA levels in the tissues: Check
the staining of Methyl Green/Pyronin Y and 28S rRNA
probe as a positive control.
- The concentration of the antisense probe: Raise the concentration of the antisense probe.
- The concentration of proteinase K : Change the concentration of proteinase K (e.g. a lower or higher concentration).
- The optimal concentration of the antibody: Carry out the
titration of the antibody using immunodetection method
to verify whether the probes are optimally labeled.
- If you use direct enzyme immunohistochemistry as visualization system, you may change it to indirect method.
• With the sense and/or antisense probes, background staining
is found.
- Stringent washing: Use a more stringent washing at lower
concentrations of salt (e.g. 0.5 x SSC, 0.2 x SSC).
- Hybridization temperature: Raise the temperature about
5°C.
- Hybridization mixtures: Add heparin and SDS to the mixture.
References
Adams JC (1981) Heavy metal intensification of DAB-based HRP reaction
products. J Histochem Cytochem 29:775
Feavers 1M, McEwan II, Liang H, Jost J-p (1989) An estradiol-dependent
protein from chicken liver binds single-stranded DNA and RNA. J
Bio Chern 264:9114-9117
Franklin WA, Lo KM, Haseltine WA (1982) J BioI Chern 257:13535-13543
Kimelman D, Kirschner MW (1989) An antisense mRNA directs the covalent modification of the transcript encoding fibroblast growth factor in
Xenopus oocytes. Cell 59:687-696
YOSHITAKA HISHIKA '" A ET AL.
• To determine the optimal condition of in situ hybridization,
positive control probe should be used.
T rou bleshooti ng
• With the antisense probe, none or weak staining is found.
- Inadequate amount of mRNA levels in the tissues: Check
the staining of Methyl Green/Pyronin Y and 28S rRNA
probe as a positive control.
- The concentration of the antisense probe: Raise the concentration of the antisense probe.
- The concentration of proteinase K : Change the concentration of proteinase K (e.g. a lower or higher concentration).
- The optimal concentration of the antibody: Carry out the
titration of the antibody using immunodetection method
to verify whether the probes are optimally labeled.
- If you use direct enzyme immunohistochemistry as visualization system, you may change it to indirect method.
• With the sense and/or antisense probes, background staining
is found.
- Stringent washing: Use a more stringent washing at lower
concentrations of salt (e.g. 0.5 x SSC, 0.2 x SSC).
- Hybridization temperature: Raise the temperature about
5°C.
- Hybridization mixtures: Add heparin and SDS to the mixture.
References
Adams JC (1981) Heavy metal intensification of DAB-based HRP reaction
products. J Histochem Cytochem 29:775
Feavers 1M, McEwan II, Liang H, Jost J-p (1989) An estradiol-dependent
protein from chicken liver binds single-stranded DNA and RNA. J
Bio Chern 264:9114-9117
Franklin WA, Lo KM, Haseltine WA (1982) J BioI Chern 257:13535-13543
Kimelman D, Kirschner MW (1989) An antisense mRNA directs the covalent modification of the transcript encoding fibroblast growth factor in
Xenopus oocytes. Cell 59:687-696