Nuclear DNA content determination of paraffin-embedded liver tissue using flow cytometry.

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We described a method for flow cytometric determination of nuclear DNA content of paraffinembedded liver tissue and discussed a potential pitfall of analysis of DNA histogram from this method in comparison with that from detergent-trypsin method which had been devised for frozen materials.<BR>In order to obtain the best quality of DNA histogram from paraffin-embedded liver tissue, it was necessary for sample preparation to be carried out as follows. Ten percent formalin fixed and paraffinembedded liver tissue was cut at 50μm thickness. Sections were dewaxed with xylene and were rehydrated through a series of ethanol. Single nuclear suspensions were prepared by incubation in 1.0% pepsin, pH1.5, at 37°C for 90 to 120 min with intermittent vortexing. After that, nuclei were treated with RNase at 37°C for 30 min and were stained with propidium iodide.<BR>It was demonstrated that DNA aneuploidy could be detected from paraffin-embedded materials as well as from frozen materials. HoWever, since there were many nuclear fragments in a sample from paraffin-embedded materials, there was a possibility that S-phase fraction was overestimated.
社団法人日本肝臓学会の論文