Simultaneous ultrastructural analysis of fluorochrome-photoconverted diaminobenzidine and gold immunolabelling in cultured cells

Eur J Histochem. 2013 Sep 16;57(3):e26. doi: 10.4081/ejh.2013.e26.

Abstract

Diaminobenzidine photoconversion is a technique by which a fluorescent dye is transformed into a stably insoluble, brown, electrondense signal, thus enabling examination at both bright field light microscopy and transmission electron microscopy. In this work, a procedure is proposed for combining photoconversion and immunoelectron microscopy: in vitro cell cultures have been first submitted to photoconversion to analyse the intracellular fate of either fluorescent nanoparticles or photosensitizing molecules, then processed for transmission electron microscopy; different fixative solutions and embedding media have been used, and the ultrathin sections were finally submitted to post-embedding immunogold cytochemistry. Under all conditions the photoconversion reaction product and the target antigen were properly detected in the same section; Epon-embedded, osmicated samples required a pre-treatment with sodium metaperiodate to unmask the antigenic sites. This simple and reliable procedure exploits a single sample to simultaneously localise the photoconversion product and a variety of antigens allowing a specific identification of subcellular organelles at the ultrastructural level.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3,3'-Diaminobenzidine / chemistry
  • Animals
  • Cell Line
  • Cells / ultrastructure*
  • Cells, Cultured
  • Fluorescent Dyes / chemistry*
  • Gold / chemistry*
  • HeLa Cells
  • Humans
  • Immunohistochemistry*
  • Microscopy, Electron, Transmission
  • Rats

Substances

  • Fluorescent Dyes
  • 3,3'-Diaminobenzidine
  • Gold