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

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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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Manuela Costanzo, Manuela Malatesta (2023)
Histochemistry of Single Molecules. Methods in Molecular Biology, 2566, 333.
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M. Malatesta, V. Galimberti, B. Cisterna, M. Costanzo, M. Biggiogera, C. Zancanaro (2014)
Chitosan nanoparticles are efficient carriers for delivering biodegradable drugs to neuronal cells. Histochemistry and Cell Biology, 141(5), 551.
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M. Malatesta, C. Pellicciari, B. Cisterna, M. Costanzo, V. Galimberti, M. Biggiogera, C. Zancanaro (2014)
Tracing nanoparticles and photosensitizing molecules at transmission electron microscopy by diaminobenzidine photo-oxidation. Micron, 59, 44.
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Natalia H. Revelo, Silvio O. Rizzoli (2016)
The Membrane Marker mCLING Reveals the Molecular Composition of Trafficking Organelles. Current Protocols in Neuroscience, 74(1).
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Manuela Malatesta (2021)
Histochemistry for nanomedicine: Novelty in tradition. European Journal of Histochemistry, 65(4).
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Matteo Pedrazzoli, Manuela Medelin, Federica Marchiotto, Barbara Cisterna, Manuela Malatesta, Mario Buffelli (2021)
An improved and simplified protocol to combine Golgi-Cox staining with immunofluorescence and transmission electron microscopy techniques. Neurochemistry International, 142, 104922.
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Manuela Malatesta (2021)
Transmission Electron Microscopy as a Powerful Tool to Investigate the Interaction of Nanoparticles with Subcellular Structures. International Journal of Molecular Sciences, 22(23), 12789.

Supporting Agencies

Fondazione Cariverona, project Verona Nanomedicine Initiative

How to Cite



Simultaneous ultrastructural analysis of fluorochrome-photoconverted diaminobenzidine and gold immunolabeling in cultured cells. (2013). European Journal of Histochemistry, 57(3), e26. https://doi.org/10.4081/ejh.2013.e26