Kanai Y, Tanuma S, Sugimura T
Proc Natl Acad Sci U S A. 1981 May;78(5):2801-4. doi: 10.1073/pnas.78.5.2801.
Randomly and synchronously growing HeLa cells were tested for poly(ADP-ribose) by direct and indirect immunofluorescent antibody techniques. Fluorescence of poly(ADP-ribose) was seen only in the nuclei of intact cells when the direct immunofluorescent antibody technique was used but in both the nuclei and cytoplasm when the indirect immunofluorescent antibody technique was used; fluorescence in the cytoplasm was nonspecific. When randomly or synchronously growing HeLa cells were fixed in acetone and treated with DNase I before incubation with fluorescein-labeled antibody, intense fluorescence was observed only in the nuclei when the direct immunofluorescent staining technique was used. Addition of 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase, with the DNase I completely abolished the fluorescence in the nuclei of synchronously and randomly growing HeLa cells, except in M-phase nuclei. These results suggest that poly(ADP-ribose) can be synthesized even in the nuclei of acetone-fixed HeLa cells from "endogenous NAD+" during incubation with fluorescent antibody and also that the fluorescence of chromosomes of HeLa cells in the M phase is, in fact, due to the in situ presence of poly(ADP-ribose), not to poly(ADP-ribose) synthesized during incubation with antibody.
通过直接和间接免疫荧光抗体技术，对随机同步生长的HeLa细胞进行聚（ADP - 核糖）检测。当使用直接免疫荧光抗体技术时，聚（ADP - 核糖）的荧光仅在完整细胞的细胞核中可见，但使用间接免疫荧光抗体技术时，细胞核和细胞质中均可见；细胞质中的荧光是非特异性的。当随机或同步生长的HeLa细胞用丙酮固定，并在与荧光素标记抗体孵育前用DNase I处理时，使用直接免疫荧光染色技术仅在细胞核中观察到强烈荧光。在DNase I中加入聚（ADP - 核糖）聚合酶的有效抑制剂3 - 氨基苯甲酰胺，完全消除了同步和随机生长的HeLa细胞细胞核中的荧光，但M期细胞核除外。这些结果表明，在与荧光抗体孵育期间，即使在丙酮固定的HeLa细胞的细胞核中，也可以从“内源性NAD +”合成聚（ADP - 核糖），并且HeLa细胞M期染色体的荧光实际上是由于聚（ADP - 核糖）的原位存在，而不是与抗体孵育期间合成的聚（ADP - 核糖）。
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