Comparison of treatment regimens to sensitize in situ hybridization for low-abundance calmodulin transcripts in the white matter of the rat spinal cord

Csaba Szigeti, Beatrix Kovacs, Elod Kortvely, Karoly Gulya

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

We compared two in situ hybridization protocols for the detection of low-abundance calmodulin (CaM) I mRNA populations in the lipid-rich white matter of the rat lumbar spinal cord. Digoxigenin-labeled, CaM I gene-specific antisense and sense cRNA probes were used in these experiments. Although microwave heating followed by chloroform and 0.1% triton X-100 treatment resulted in the specific labeling of several cells in the gray matter of the spinal cord with a low nonspecific signal, it did not detect any CaM I expressing cells in the white matter. The protocol involving a hybridization solution adjusted to a slightly alkaline pH (pH 8.0), however, resulted in the detection of a large number of CaM-expressing cells not only in the gray matter, but also in the white matter of the spinal cord, with a nonspecific hybridization signal that was essentially identical to that of the background, and it also retained a much better overall tissue quality as compared with the protocol involving microwave heating and triton X-100 treatment. Numerous medium-sized astrocyte-like cells and smaller cells resembling oligodendrocytes were detected that expressed CaM I mRNAs throughout the white matter of the lumbar spinal cord. Thus, in situ hybridization carried out at a slightly alkaline pH is a far superior method for the detection of low-abundance mRNA populations in lipid-rich regions of the central nervous system, such as the white matter areas, as compared with microwave heating combined with triton X-100 treatment.

Original languageEnglish
Pages (from-to)1-6
Number of pages6
JournalActa Biologica Szegediensis
Volume47
Issue number1-4
Publication statusPublished - Dec 1 2003

    Fingerprint

Keywords

  • Alkaline pH
  • Calmodulin
  • In situ hybridization
  • Microwave
  • Rat
  • Spinal cord
  • Triton X-100
  • White matter

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this