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

  • M. Malatesta | manuela.malatesta@univr.it Dipartimento di Scienze Neurologiche e del Movimento, sezione di Anatomia e Istologia, Università di Verona, Italy.
  • C. Zancanaro Dipartimento di Scienze Neurologiche e del Movimento, sezione di Anatomia e Istologia, Università di Verona, Italy.
  • M. Costanzo Dipartimento di Scienze Neurologiche e del Movimento, sezione di Anatomia e Istologia, Università di Verona, Italy.
  • B. Cisterna Dipartimento di Scienze Neurologiche e del Movimento, sezione di Anatomia e Istologia, Università di Verona, Italy.
  • C. Pellicciari Dipartimento di Biologia e Biotecnologie "L. Spallanzani", Laboratorio di Biologia cellulare e Neurobiologia, Università di Pavia, Italy.

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.

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Published
2013-09-16
Section
Technical Notes
Supporting Agencies
Fondazione Cariverona, project Verona Nanomedicine Initiative
Keywords:
diaminobenzidine, photoconversion, immunogold cytochemistry, transmission electron microscopy.
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How to Cite
Malatesta, M., Zancanaro, C., Costanzo, M., Cisterna, B., & Pellicciari, C. (2013). Simultaneous ultrastructural analysis of fluorochrome-photoconverted diaminobenzidine and gold immunolabeling in cultured cells. European Journal of Histochemistry, 57(3), e26. https://doi.org/10.4081/ejh.2013.e26