COMBINATION OF FEULGEN NUCLEAR REACTION WITH IMMUNOFLUORESCENT STAINING FOR PHOTOPRODUCTS OF DNA AFTER UV-IRRADIATION

Publisher Website
Google Scholar
Abstract
Immunofluorescent staining for nuclear UV photoproducts was combined with nuclear Feulgen reaction for cytofluorometry to quantitate the potency of excision repair of the lesions per unit amount of DNA on a single cell. Isolated hepatocytes and cerebellar neurons of adult rat were UV-irradiated and reacted with rabbit antiserum containing antibodies to photoproducts of DNA after UV-irradiation. The UV induced lesions were demonstrated by indirect immunofluorescent technique using FITC [first important soluble component] labeled anti-rabbit''s immunoglobulin antibody. The immunofluorescent staining was combined with subsequent Feulgen nuclear reaction after stabilizing the fluorescent dye-immunoglubulin complex by post-fixation with absolute ethanol. As a linear relationship was found between fluorescence intensity and concentration of free and protein-bound FITC up to absorbance value of 0.1, intensity of the immunofluorescent staining was controlled not to exceed this limit. Two peaks of fluorescence of FITC (520 nm) and Feulgen dye (613 nm) were separately identified on a double-stained nucleus with respective excitation by 450 nm and 543 nm without interference of each other''s fluorescence. Proportionality between DNA content and amount of Feulgen fluorescence was preserved in the double stained hepatocytes and cerebellar neurons. Linear relationships were noted between the amounts of Feulgen DNA and nuclear FITC in the same cells immediately after UV-irradiation. The amounts of photoproducts produced in unit amount of nuclear DNA are equal in all cells irrespective of the kinds of tissues and stages of cell cycle.

This publication has 1 reference indexed in Scilit: