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(American Journal of Pathology. 2003;162:381-389.)
© 2003 American Society for Investigative Pathology

Technical Advance

In Situ Hybridization AT-Tailing with Catalyzed Signal Amplification for Sensitive and Specific in Situ Detection of Human Immunodeficiency Virus-1 mRNA in Formalin-Fixed and Paraffin-Embedded Tissues

Noriko Nakajima*, Petronela Ionescu*{dagger}, Yuko Sato*, Michie Hashimoto*, Toshihiro Kuroita{ddagger}, Hidehiro Takahashi*, Hiroshi Yoshikura* and Tetsutaro Sata*

From the Department of Pathology,* National Institute of Infectious Diseases, Tokyo, Japan; the V. Babes Infectious Diseases Hospital,{dagger} Bucharest, Romania; and Toyobo,{ddagger} Tsuruga, Fukui, Japan

In situ hybridization is one of the most important techniques to visualize gene expression at the cellular level in various tissues. The in situ hybridization-AT tailing (ISH-AT) method uses a specially designed and synthesized oligonucleotide probe that has (AT)10 on the 3' side. This (AT)10 of the probe is elongated by {Delta}Tth DNA polymerase in the presence of dATP, dTTP, and labeled dUTP in the tissue after hybridization. Through this process the target is labeled with many hapten molecules. In this study, we detected human immunodeficiency virus type 1 RNA in formalin-fixed and paraffin-embedded tissues obtained from autopsied patients with acquired immunodeficiency syndrome by combining ISH-AT with the catalyzed signal amplification (CSA) system (ISH-AT-CSA), although we failed to detect signals from the same samples by conventional in situ hybridization using RNA probes (RISH) with CSA (RISH-CSA). We demonstrated that the ISH-AT-CSA method was superior to RISH-CSA in terms of both sensitivity and specificity, and that it was applicable to fluorescence in situ hybridization and double staining with immunohistochemistry for the characterization of cell phenotypes.




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