Cytofluorometry on cells isolated from paraffin sections after blocking of the background fluorescence by azocarmin G

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Abstract
A technique for isolation of cells from paraffin embedded tissue is indispensable for the performance of Feulgen-DNA cytofluorometry in parallel with the definition of histological characteristics. Background fluorescence due to nonspecific dye-binding by a “pseudo-plasmal reaction” is usually found to be so intense on cells isolated from formalin-fixed tissues, that we are often forced to abandon quantitative DNA determinations. In the present work, we report that fixation of tissues with Carnoy's fixative for 12 h at 5° C not only reduces nonspecific dye-binding but also facilitates the process of cell isolation. Furthermore, we find that pre-treatment of cells isolated from Carnoy-fixed tissues with acidic azocarmin G solution completely blocks nonspecific dye-binding in subsequent acriflavine Feulgen nuclear staining. This combination of techniques for specimen preparation enables us to carry out Feulgen-DNA cytofluorometry on cells isolated from histological sections with satisfactorily low coefficients of variation (less than 8%). The techniques should be widely applicable for parallel DNA determinations and histology.