A DOUBLE LABELING TECHNIQUE FOR PERFORMING IMMUNOCYTOCHEMISTRY AND INSITU HYBRIDIZATION IN VIRUS-INFECTED CELL-CULTURES AND TISSUES
A DOUBLE LABELING TECHNIQUE FOR PERFORMING IMMUNOCYTOCHEMISTRY AND INSITU HYBRIDIZATION IN VIRUS-INFECTED CELL-CULTURES AND TISSUES
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DOI：
10.1016/0166-0934(85)90033-3
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发表时间：
1985-01-01
影响因子：
3.1
通讯作者：
CLEMENTS, JE
中科院分区：
文献类型：
--
作者：
GENDELMAN, HE;MOENCH, TR;CLEMENTS, JE
本报告描述了一种免疫细胞化学和原位杂交相结合的方法，该方法可使细胞或病毒抗原以及同一细胞中的病毒RNA可视化。用维斯纳病毒或麻疹病毒感染的培养物用高碘酸 - 赖氨酸 - 多聚甲醛 - 戊二醛固定，使用针对病毒或细胞蛋白的抗体通过亲和素 - 生物素 - 过氧化物酶技术染色，然后与放射性标记的特异性DNA探针一起孵育（原位杂交）。免疫过氧化物酶染色在杂交过程中得以保留。通过在杂交溶液和杂交后洗涤液中加入0.1%的Triton X - 100，防止了探针在过氧化物酶染色细胞上的非特异性附着。与未染色细胞的杂交相比，过氧化物酶染色细胞上的原位杂交信号（每个细胞的银颗粒数）有所降低。这种双重标记技术还应用于感染维斯纳病毒的绵羊和感染麻疹病毒HNT株的小鼠的石蜡包埋组织切片。在免疫细胞化学鉴定的滑膜巨噬细胞中检测到维斯纳病毒RNA。这些细胞中含病毒RNA的数量比含病毒蛋白的数量多。在麻疹病毒感染的大脑中，仅在含病毒蛋白的细胞中检测到病毒RNA。该技术通过以下方式为病毒发病机制的研究提供了一种新途径：确定宿主中被感染的细胞类型以及确定持续性感染过程中病毒生命周期的阻断点。
This report describes a combined immunocytochemical and in situ hybridization procedure which allows visualization of cellular or viral antigens and viral RNA in the same cell. Cultures infected with visna or measles virus were fixed in periodate-lysine-paraformaldehyde-glutaraldehyde, stained by the avidin-biotin-peroxidase technique using antibodies to viral or cellular proteins and then incubated with radiolabeled specific DNA probes (in situ hybridization). The immunoperoxidase stain was preserved through the hybridization procedure. Nonspecific sticking of probes over peroxidase stained cells was prevented by incorporation of 0.1% Triton X-100 into the hybridization solution and the post-hybridization washes. The in situ hybridization signal (silver grains/cell) on peroxidase-stained cells was reduced relative to hybridization with unstained cells. The double labeling technique was also applied to sections of paraffin-embedded tissues from a sheep infected with visna virus and mice infected with the HNT strain of measles virus. Visna virus RNA was detected in immunocytochemically identified macrophages in the synovium. A greater number of these cells had viral RNA than had viral protein. In measles virus-infected brains viral RNA was detected only in cells with viral protein. This technique provides a new approach to the study of viral pathogenesis by: identifying the types of cells which are infected in the host and identifying points of blockade in the virus life cycle during persistent infections.