Fluorescent cytochemical detection of sialidase activity using 5-bromo-4-chloroindol-3-yl-α-D-N-acetylneuraminic acid as the substrate

Megumi Saito, Hitoshi Hagita, Yoshiharu Iwabuchi, Ikuo Fujii, Kiyoshi Ikeda, Masaki Ito

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)

Abstract

A novel fluorescent cytochemical method for sialidase activity was developed using 5-bromo-4-chloroindol-3-yl-α-D-N-acetylneuraminic acid (X-Neu5Ac) as the substrate. Intact nuclei were isolated from porcine liver and incubated at 37°C for 3 h with 1 mM X-Neu5Ac at pH 4.8. The nuclei were stained with blue color that was derived from the oxidized compound of the reaction product X (5-bromo-4-chloro-3-hydroxyindole). A specific sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetyl-neuraminic acid, suppressed the staining in a dose-dependent manner. Despite the specificity of the cytochemical reaction, the staining was too weak to analyze the staining distribution and pattern of individual nuclei. To attain more sensitive detection of sialidase activity, the nuclei were incubated with X-Neu5Ac in the presence of Fast Red Violet LB. Individual nuclei of porcine liver were clearly stained with fluorescence that was produced by the conjugated compound of product X with Fast Red Violet LB. This fluorescent cytochemical method was also employed successfully for detection of sialidase activity of intact GOTO neuroblastoma cells in culture. The present method should provide a useful tool for investigating the localization and stage-specific expression of sialidase activity in tissues and cells.

Original languageEnglish
Pages (from-to)453-458
Number of pages6
JournalHistochemistry and Cell Biology
Volume117
Issue number5
DOIs
Publication statusPublished - 2002 Jun 8
Externally publishedYes

Keywords

  • Cell nucleus
  • Neuroblastoma cells
  • Porcine liver
  • Sialidase
  • X-Neu5Ac

ASJC Scopus subject areas

  • Histology
  • Molecular Biology
  • Medical Laboratory Technology
  • Cell Biology

Fingerprint Dive into the research topics of 'Fluorescent cytochemical detection of sialidase activity using 5-bromo-4-chloroindol-3-yl-α-D-N-acetylneuraminic acid as the substrate'. Together they form a unique fingerprint.

Cite this