Selective binding of nucleotide probes by eosinophilic cationic protein during in situ hybridisation

Richard D. Ellis, Paul Ashwood, Jonathan J. Powell, Paul D. Taylor, Richard Poulsom, Richard P H Thompson, Neville A. Punchard

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

During in situ hybridisation on frozen and paraffin-embedded sections of bowel for IκBα, oligodeoxyribonucleotide probes were found to bind more avidly to eosinophils than target mRNA. This binding could not be obviated using strategies previously employed to block either binding of long DNA probes (200-mers) to eosinophils in bone marrow smears, or of riboprobes to eosinophils in sections of bowel, without removing specific hybridisation of probes. That this binding could arise through interaction of anionic oligodeoxyribonucleotides with eosinophil cationic protein, which has an unusually high pI, and is abundant in cytoplasmic granules of eosinophils, was demonstrated in vitro using real-time biomolecular interaction analysis with a BiacoreX instrument. Finally, a relationship between probe hydrophobicity, measured by reverse phase ion-pair high performance liquid chromatography, and in situ binding of individual probes to eosinophils was demonstrated. Effective tissue penetration by hydrophobic probes and subsequent strong probe-eosinophilic cationic protein interactions therefore may confound the interpretation of in situ hybridisation performed with oligonucleotide probes in eosinophil-containing tissues, such as bowel and nasal polyps.

Original languageEnglish (US)
Pages (from-to)153-160
Number of pages8
JournalHistochemical Journal
Volume34
Issue number3-4
DOIs
StatePublished - Mar 2002
Externally publishedYes

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ASJC Scopus subject areas

  • Cell Biology
  • Anatomy

Cite this

Ellis, R. D., Ashwood, P., Powell, J. J., Taylor, P. D., Poulsom, R., Thompson, R. P. H., & Punchard, N. A. (2002). Selective binding of nucleotide probes by eosinophilic cationic protein during in situ hybridisation. Histochemical Journal, 34(3-4), 153-160. https://doi.org/10.1023/A:1020942531028