title: 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.
abstract: 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 degrees C) and more standard low (4 degrees C) 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 degrees C were utilized to determine the optimal hybridization conditions for detection of SIV productively infected cells. Fixation conditions of 40 degrees C and hybridization conditions of 50-55 degrees C 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.
authors.affiliation: Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Beth A
authors.initials: BA
authors.lastname: Fallert
authors.forename: Todd A
authors.initials: TA
authors.lastname: Reinhart
mesh_terms: D000818:Animals; D005557:Formaldehyde; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D008198:Lymph Nodes / Q000821:virology; D008253:Macaca mulatta; D015347:RNA Probes; D012367:RNA, Viral / Q000032:analysis*; D016097:Simian Acquired Immunodeficiency Syndrome / Q000821:virology*; D015302:Simian Immunodeficiency Virus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D013696:Temperature; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D015347:RNA Probes; D012367:RNA, Viral; D005557:Formaldehyde