Ultrastructural localization of herpes simplex virus RNA by in situ hybridization.
Ultrastructural localization of herpes simplex virus RNA by in situ hybridization.
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通过原位杂交对单纯疱疹病毒 RNA 进行超微结构定位。
DOI：
10.1177/37.1.2535704
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发表时间：
1989
期刊：
影响因子：
--
通讯作者：
H. F. Maassab
中科院分区：
文献类型：
--
作者：
R. Wolber;T. Beals;H. F. Maassab
我们用胶体金作为电子致密标记，用包埋前原位杂交的方法研究了病毒编码的RNA的亚细胞定位。将感染单纯疱疹病毒(HSV)的成纤维细胞固定、渗透，然后在有利于DNA-RNA杂交化的条件下与生物素标记的HSV DNA探针杂交。用5 nm链霉亲和素-金标记物定位HSV探针。透射电子显微镜显示HSV感染细胞内的5 nm金呈簇状和单线状分布。福尔马林固定的细胞平均每个胞质有4.6个簇，每个核有13.2个簇。福尔马林-戊二醛联合固定使每个细胞质和核的平均簇数分别增加到7.2个(增加57%)和17.5个(增加33%)。金簇通常位于核膜附近，但不与病毒核衣壳或内质网结合。标记不受细胞杂交前DNA酶处理的影响。核糖核酸酶消除87%的胞质和97%的核团。这些发现表明，聚集的金颗粒标记了病毒RNA，可能与多个DNA探针分子和/或金颗粒结合到RNA链上。这种新的包埋前技术可能是一种有用的工具，用于病毒-宿主细胞相互作用的超微结构评估。
We studied the subcellular localization of virally encoded RNA by pre-embedding in situ hybridization, using colloidal gold as an electron-dense marker. Fibroblasts infected with Herpes simplex virus (HSV) were fixed, permeabilized, then hybridized with a biotinylated HSV DNA probe under conditions favoring DNA-RNA hybrid formation. HSV probe was localized with 5-nm streptavidin-gold conjugates. Transmission electron microscopy revealed 5-nm gold in clusters and singlets within HSV-infected cells. Formalin-fixed cells contained a mean of 4.6 clusters per cytoplasmic profile and 13.2 clusters per nuclear profile. Combined formalin-glutaraldehyde fixation increased the mean number of clusters per cytoplasmic and nuclear profile to 7.2 (57% increase) and 17.5 (33% increase), respectively. Gold clusters were frequently located in regions adjacent to the nuclear envelope but were not bound to viral nucleocapsids or endoplasmic reticulum. Labeling was unaffected by pre-hybridization DNAse treatment of cells. RNAse eliminated 87% of cytoplasmic and 97% of nuclear clusters. These findings indicate that clustered gold particles labeled viral RNA, with probable binding of multiple DNA probe molecules and/or gold particles to RNA strands. This novel pre-embedding technique may be a useful tool for ultrastructural evaluation of virus-host cell interactions.
用于检测肌动蛋白基因表达的原位杂交方法的定量分析。
影响因子：
14.9
作者：
Lawrence,JB;Singer,RH
通讯作者：
Singer,RH
DOI：
10.1073/pnas.78.11.6633
发表时间：
1981-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
LANGER, PR;WALDROP, AA;WARD, DC
通讯作者：
WARD, DC
DOI：
10.1073/pnas.80.13.4045
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
LEARY, JJ;BRIGATI, DJ;WARD, DC
通讯作者：
WARD, DC