Extraction of nuclei from selected regions in paraffin-embedded tissue

A method for the extraction of nuclei from selected regions in paraffin-embedded tissue is described. Fifty-micrometer sections are cut, dewaxed, and rehydrated. For the final handling, the sections are manually transferred from one tray to another. The sections are put on a slide under a dissection microscope and the region of interest is isolated by scraping off the irrelevant region with a scalpel. An optimal number of single nuclei is obtained by incubation in a protease solution with intermediate syringing. The nuclei are washed and can be used for flow cytometry. Resuspension of the nuclei in foetal calf serum and cytocentrifugation results in preparations suited for image analysis. DNA cytometric measurements of nuclei in a carcinoma in situ and an invasive carcinoma region in breast tissue present in the same tissue block and in a severe dysplasia/carcinoma in situ (CIN III) region of the cervix are presented.