Efficiency of in situ hybridization as a function of probe size and fixation technique

Thomas R. Moench, Howard E. Gendelman1, Janice E. Clements, Opendra Narayan, Diane E. Griffin

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

In an attempt to improve fixation technique for viral RNA detection by in situ hybridization, we have quantitatively compared the hybridization signal obtained when measles virus or visna virus infected cell cultures were fixed with eight different fixatives and hybridized with 35S-labeled virus-complementary DNA probes of several size ranges. Small probes (mean length, 70 bases) gave higher signals than larger probes (mean lengths 140, 350, and 780 bases) with all fixatives. This increase in signal was minimal with acetic ethanol or formalin, but was dramatic with fixatives containing glutaraldehyde; with these fixatives the signals with small probes were 6.5- to 22-fold greater than with large probes. The highest signals were obtained with periodate-lysine-paraformaldehyde-glutaraldehyde (PLPG) fixed cells hybridized with small probes, and were 1.5- to 6.7-fold greater than those obtained with the commonly used fixative acetic ethanol. PLPG and other glutaraldehyde based fixatives also greatly improved the preservation of cellular morphology compared to acetic ethanol.

Original languageEnglish (US)
Pages (from-to)119-130
Number of pages12
JournalJournal of Virological Methods
Volume11
Issue number2
DOIs
StatePublished - Jun 1985

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Fixatives
In Situ Hybridization
Glutaral
Acetic Acid
Ethanol
Visna-maedi virus
Measles virus
DNA Probes
Viral RNA
Formaldehyde
Complementary DNA
Cell Culture Techniques
Viruses

Keywords

  • in situ hybridization fixation periodate-lysine-paraformaldehyde-glutaraldehyde

ASJC Scopus subject areas

  • Virology

Cite this

Efficiency of in situ hybridization as a function of probe size and fixation technique. / Moench, Thomas R.; Gendelman1, Howard E.; Clements, Janice E.; Narayan, Opendra; Griffin, Diane E.

In: Journal of Virological Methods, Vol. 11, No. 2, 06.1985, p. 119-130.

Research output: Contribution to journalArticle

Moench, Thomas R. ; Gendelman1, Howard E. ; Clements, Janice E. ; Narayan, Opendra ; Griffin, Diane E. / Efficiency of in situ hybridization as a function of probe size and fixation technique. In: Journal of Virological Methods. 1985 ; Vol. 11, No. 2. pp. 119-130.
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