Puvion-Dutilleul F
Groupe de Laboratoires de l'Institut de Recherches Scientifiques sur le Cancer, CNRS (UPR272), 94801 Villejuif, France.
J Histochem Cytochem. 1991 May;39(5):669-80. doi: 10.1177/39.5.1707905.
Highly phosphorylated proteins in situ in sections of Lowicryl-embedded cells are preferentially stained by bismuth, provided that the reactivity of the amino groups is blocked by glutaraldehyde fixation. This study showed that bismuth staining can be preceded by indirect immunocytochemistry using gold particles as markers. As a result, both immunostained and bismuth-stained proteins can be detected concomitantly on the same section. This was also carried out on sections of formaldehyde-fixed cells which were immunolabeled, then post-fixed with glutaraldehyde, and finally exposed to bismuth stain. These procedures were applied to sections of adenovirus Type 5-infected HeLa cells. Bismuth ions and viral anti-72 KD antibody bound concomitantly to intranuclear virus-induced single-stranded DNA (ssDNA) accumulation sites, structures in which viral replicative activity is intermittent, and also to the fibrillogranular peripheral replicative zones which surround the ssDNA accumulation sites and in which replication of viral genomes is continuous. The delicate fibrillar network enclosed within virus-induced compact rings of unknown function is slightly bismuth stained and binds few antibodies to viral 72 KD protein. Three intranuclear structures were stained exclusively with bismuth: the fibrillar component of the nucleolus, which is involved in ribosome formation; the interchromatin granules; and the virus-induced "fibrillar spots" of unknown significance. Thus, not all highly phosphorylated proteins in adenovirus-infected cells are viral 72 KD protein. In glutaraldehyde-fixed Miller spreads of nucleic acid molecules from adenovirus-infected cells, bismuth deposits occurred over unique thick filaments, the only portion of the viral deoxyribonucleoprotein molecules shown to be associated with viral 72 KD protein. In vitro studies revealed that the latter protein, known to be multiply phosphorylated, concomitantly binds anti-72 KD antibody and bismuth ions. These data have broadened the scope of the use of bismuth staining. Taken together, they indicate that in adenovirus infection highly phosphorylated proteins accumulate over intranuclear structures related to both replication of viral genomes and alteration of ribosomal metabolism.
在用Lowicryl包埋的细胞切片中，高度磷酸化的蛋白质原位优先被铋染色，前提是氨基的反应性被戊二醛固定所阻断。本研究表明，铋染色可以先进行间接免疫细胞化学，使用金颗粒作为标记物。结果，可以在同一切片上同时检测到免疫染色和铋染色的蛋白质。这也在甲醛固定的细胞切片上进行，这些细胞先进行免疫标记，然后用戊二醛后固定，最后用铋染色。这些程序应用于5型腺病毒感染的HeLa细胞切片。铋离子和病毒抗72 KD抗体同时结合到核内病毒诱导的单链DNA（ssDNA）积累位点，病毒复制活性间歇性的结构，也结合到围绕ssDNA积累位点的纤维颗粒状外周复制区，病毒基因组在其中持续复制。包含在功能未知的病毒诱导的紧密环内的精细纤维网络被铋轻微染色，并且很少结合病毒72 KD蛋白的抗体。有三种核内结构仅被铋染色：参与核糖体形成的核仁纤维成分；染色质间颗粒；以及意义未知的病毒诱导的“纤维斑点”。因此，腺病毒感染细胞中的并非所有高度磷酸化蛋白质都是病毒72 KD蛋白。在戊二醛固定的腺病毒感染细胞核酸分子的Miller铺展片中，铋沉积物出现在独特的粗丝上，这是病毒脱氧核糖核蛋白分子中唯一显示与病毒72 KD蛋白相关的部分。体外研究表明，已知该蛋白被多次磷酸化，同时结合抗72 KD抗体和铋离子。这些数据拓宽了铋染色的应用范围。综合来看，它们表明在腺病毒感染中高度磷酸化蛋白质在与病毒基因组复制和核糖体代谢改变相关的核内结构上积累。
