Cytofluorometry on cells isolated from paraffin sections after blocking of the background fluoroscence by azocarmine G.
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 the fixation of tissues with Carnoy's fixative for 12 h at 5 degrees 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.
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D004396:Coloring Agents; D004247:DNA / Q000032:analysis*; D005404:Fixatives; D005470:Fluorometry / Q000379:methods*; D006651:Histocytochemistry; D010232:Paraffin; D010619:Phenazines; D051381:Rats; D012394:Rosaniline Dyes
D004396:Coloring Agents; C007722:Feulgen stain; D005404:Fixatives; D010619:Phenazines; D012394:Rosaniline Dyes; C025171:azocarmine G; D010232:Paraffin; D004247:DNA