Immunocytometric determination of the alpha and beta isoforms of human DNA topoisomerase II: influence of different fixatives.
The immunoreactivity of the alpha and beta isoforms (170 and 180 kDa, respectively) of human DNA topoisomerase II (topo II) toward specific monoclonal antibodies (MoAbs), was investigated in HeLa cells. The extent of antigen preservation achieved with the dehydrating fixatives, ethanol and acetone, was compared with that obtained with the cross-linking agent paraformaldehyde. Binding of each MoAb to the relative isozyme was detected by indirect immunofluorescence labelling and quantified by flow cytometry. The amount of antigen detectable was calculated by normalizing the mean fluorescence intensity of samples incubated with specific MoAbs, to the respective background fluorescence. For both isozymes, acetone provided the highest immunofluorescence/background ratio. A strong reduction in the immunoreactivity of the 180-kDa isoform was observed after ethanol, and to a minor extent after paraformaldehyde fixation, while reactivity of the 170-kDa isoform was not significantly affected. Cell cycle distribution of each protein was assessed by dual-parameter analysis of immunofluorescence vs. DNA content. No evident difference in the cell cycle distribution of each isozyme was found with each fixation protocol. At the morphological level, the pattern of immunofluorescence showed that the localization of each isozyme was very similar for all these fixatives. These results confirm the great sensitivity of the 180-kDa isoform to degradation, and indicate that fixation has to be carefully chosen in order to determine the relative amount of topo II isoforms with immunocytometric techniques.
Prosperi
E
E
Dipartimento di Biologia Animale, Università di Pavia, Italy.
Negri
C
C
Bottiroli
G
G
Astaldi Ricotti
G C
GC
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002453:Cell Cycle; D004250:DNA Topoisomerases, Type II / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006652:Histological Techniques; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007527:Isoenzymes / Q000032:analysis*; D051379:Mice
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D007527:Isoenzymes; D004250:DNA Topoisomerases, Type II; D016650:Fluorescein-5-isothiocyanate