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Detection of tissue mRNA in human surgical materials by non-radioactive in situ hybridization technique may be difficult owing to inconsistent retention of tissue mRNA. This inconsistency is generally ascribed to different timing of ligation of feeding arteries during surgical operation. To improve this inconsistency, we adopted fixation in 4% paraformaldehyde/0.5% glutaraldehyde for 12-16 hr with paraffin embedding. This method resulted in improved retention of tissue mRNA: higher in sensitivity and more consistent with cases. Furthermore, this procedure has shortened the color development time of the labeled enzyme, alkaline phosphatase. This helps to reduce nonspecific reactivity because prolonged color development time (up to 24) is one of the major causes of non-specific reactions. Cut sections can be stored at 4 degrees C for more than a year, not resulting in detectable reduction of sensitivity. These are major advantages of this method particularly as human surgical specimens are concerned, because accumulation of cases is important for clinicopathologic analyses. Therefore, this fixation method is, at least, recommendable to improve in situ hybridization technique.

For Consistent Detection of Tissue mRNA in Human Surgical Material by Non-Radioactive In Situ Hybridization Technique.