Improved detection of simian immunodeficiency virus RNA by in situ hybridization in fixed tissue sections: combined effects of temperatures for tissue fixation and probe hybridization
Improved detection of simian immunodeficiency virus RNA by in situ hybridization in fixed tissue sections: combined effects of temperatures for tissue fixation and probe hybridization
复制标题
DOI：
10.1016/s0166-0934(01)00378-0
复制
发表时间：
2002-01-01
影响因子：
3.1
通讯作者：
Reinhart, TA
中科院分区：
文献类型：
--
作者：
Fallert, BA;Reinhart, TA
原位杂交检测甲醛固定的组织标本中的病毒RNA经常用于表征宿主组织内病毒复制的程度。原位测定病毒RNA表达水平的能力取决于许多因素，包括固定过程中交联的程度、用于减轻交联效应的预处理方案以及杂交和洗涤方案。为了提高我们检测恒河猴组织中猴免疫缺陷病毒（SIV）感染细胞的能力，我们根据经验测试了在4%多聚甲醛/磷酸盐缓冲盐水中非常规高温（40 ℃）和更标准低温（4 ℃）固定对原位杂交信号的影响。此外，利用37 - 75 ℃的杂交温度来确定检测SIV有效感染细胞的最佳杂交条件。40 ℃的固定条件和50-55 ℃的杂交条件被鉴定为提供检测RNA（+）细胞和定量每个细胞的信号的最大灵敏度，同时仍然允许通过免疫组织化学染色检测抗原表位。这些数据表明，病毒RNA原位杂交后的信号强度取决于组织固定和原位杂交温度的综合影响。(C)2002 Elsevier Science B. V.保留所有权利。
In situ hybridization detection of viral RNAs in formaldehyde-fixed tissue specimens is used frequently to characterize the extent of viral replication within host tissues. The ability to determine the level of expression of viral RNAs in situ is dependent upon many factors including the extent of cross-linking during fixation, the pretreatment regimen utilized to relieve the effects of cross-linking, and the hybridization and wash protocols. In efforts to improve our ability to detect cells infected productively by simian immunodeficiency virus (SIV) in rhesus macaque tissues, the effects of unconventionally high (40 degreesC and more standard low (4 degreesC temperature fixation in 4% paraformaldehyde/phosphate buffered saline were tested empirically on in situ hybridization signals. In addition, hybridization temperatures ranging between 37 and 75 degreesC were utilized to determine the optimal hybridization conditions for detection of SIV productively infected cells. Fixation conditions of 40 degreesC and hybridization conditions of 50-55 degreesC were identified as providing the greatest sensitivity for detecting RNA(+) cells and for quantitating the signal per cell, while still allowing antigenic epitopes to be detected by immunohistochemical staining. These data indicate that the signal intensity following in situ hybridization for viral RNAs is dependent upon the combined effects of tissue fixation and in situ hybridization temperatures. (C) 2002 Elsevier Science B.V. All rights reserved.