Detection protocols for biotinylated probes: Optimization using multistep techniques

S. McQuaid*, G. M. Allan

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

31 Citations (Scopus)


Recent studies using biotinylated in situ hybridization (ISH) have utilized a wide range of detection protocols for the biotinylated hybrids, leading to conflicting reports in the literature regarding sensitivity. In this study we compared 11 different detection protocols for biotinylated ISH using a measles virus-specific RNA probe on formalin-fixed, paraffin-embedded central nervous system tissue infected with measles virus. Maximum sensitivity was achieved with five-step detection protocols incorporating the use of a monoclonal antibody to biotin. Single-step detection protocols were found to be insensitive, as shown by their failure to detect viral nucleic acid in infected white-matter cells. Only by increasing the number of steps in the detection protocols were these infected cells demonstrable. Unless pre-hybridization, hybridization, and detection protocols are optimized, the results obtained in pathogenicity studies using ISH could be misinterpreted, leading to false conclusions about nucleic acid distribution. This also applies to the ever-increasing use of ISH for diagnostic purposes.

Original languageEnglish
Pages (from-to)569-574
Number of pages6
JournalJournal of Histochemistry and Cytochemistry
Issue number4
Publication statusPublished - 1992
Externally publishedYes

Bibliographical note

Copyright 2017 Elsevier B.V., All rights reserved.


  • Biotin
  • In situ hybridization
  • Monoclonal antibody
  • Optimization

ASJC Scopus subject areas

  • Anatomy
  • Histology


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