A double labeling technique for performing immunocytochemistry and in situ hybridization in virus infected cell cultures and tissues

Howard E. Gendelman, Thomas R. Moench, Opendra Narayan, Diane E. Griffin, Janice E. Clements

Research output: Contribution to journalArticle

34 Scopus citations

Abstract

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-bio-tin-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: (1) identifying the types of cells which are infected in the host and (2) identifying points of blockade in the virus life cycle during persistent infections.

Original languageEnglish (US)
Pages (from-to)93-103
Number of pages11
JournalJournal of Virological Methods
Volume11
Issue number2
DOIs
Publication statusPublished - Jun 1985

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Keywords

  • immunocytochemistry
  • in situ hybridization

ASJC Scopus subject areas

  • Virology

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