Allen R L, Olins D E
Chromosoma. 1984;91(1):82-6. doi: 10.1007/BF00286488.
A modification of the silver-staining techniques for nucleolar organizing regions (NORs) was used to stain selectively the macronuclear replication bands (RBs) and nucleoli in hypotrichous ciliated protozoa (Euplotes, Stylonychia, and Oxytricha). Silver staining of both types of structures was trypsin-sensitive and DNase I-insensitive, suggesting the involvement of proteins. Silver-staining proteins in the RB were differentially extracted with acid, without any decrease in nucleolar staining. Triton-acid-urea gel electrophoresis of an acid extract of Euplotes macronuclei revealed enhanced silver reaction with a single protein upon selective silver staining. An abundance of thiol groups was also demonstrated in the RBs and nucleoli by the fluorochrome 3-(4-maleimidylphenyl)-7-diethylamino-4-methyl coumarin (coumarin maleimide). Histochemical studies, including blocking thiols with N-ethyl maleimide (NEM), indicated that thiols were not necessary for silver staining, and that proteins in the RBs and nucleoli reacting with coumarin maleimide were not acid extractable.
一种用于核仁组织区（NORs）的银染技术经过改良，用于选择性地对腹毛目纤毛虫（真核草履虫、长吻虫和尖毛虫）中的大核复制带（RBs）和核仁进行染色。这两种结构的银染对胰蛋白酶敏感，但对DNase I不敏感，表明蛋白质参与其中。用酸对RB中的银染蛋白进行差异提取后，核仁染色没有任何减少。对真核草履虫大核的酸提取物进行Triton-酸-尿素凝胶电泳，结果显示在选择性银染后，一种单一蛋白质的银反应增强。荧光染料3-(4-马来酰亚胺基苯基)-7-二乙氨基-4-甲基香豆素（香豆素马来酰亚胺）也证明了RB和核仁中存在大量的巯基。包括用N-乙基马来酰亚胺（NEM）封闭巯基在内的组织化学研究表明，巯基对于银染不是必需的，并且与香豆素马来酰亚胺反应的RB和核仁中的蛋白质不能被酸提取。
