High-resolution FEISEM detection of DNA centromeric probes in HeLa metaphase chromosomes

E. Rizzi, M. Falconi, R. Rizzoli, B. Baratta, L. Manzoli, A. Galanzi, G. Lattanzi, G. Mazzotti

Research output: Contribution to journalArticlepeer-review

Abstract

HeLa metaphase chromosome spreads were hybridized with centromeric biotinylated DNA probes and detected with gold-conjugated anti-biotin antibodies. Chromosomes were observed by an in-lens field emission scanning electron microscope (FEISEM), which permits detection of biological samples without any coating. DNA probes were well localized in the centromeric region of chromosomes and there was clear discrimination between 10 nm fibers that hybridized to DNA probes and those that did not hybridize. This approach shows that in situ hybridization can be directly visualized at the FEISEM level by evaluating only secondary electron emission, which allows physical localization of the hybridized probe with high resolution so that backscatter detection represents only a control. Because chromosomes maintain the 10-nm fiber organization after in situ hybridization procedures, our data suggest that this fiber represents the lowest order of chromatin arrangement that permits transitory DNA denaturation.

Original languageEnglish
Pages (from-to)413-419
Number of pages7
JournalJournal of Histochemistry and Cytochemistry
Volume43
Issue number4
Publication statusPublished - 1995

Keywords

  • Chromosome fibers
  • Chromosomes
  • DNA centromeric probes
  • Field emission in-lens scanning electron microscopy
  • High- resolution in situ hybridization
  • Immunogold detection

ASJC Scopus subject areas

  • Cell Biology
  • Anatomy

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