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Journal of Histochemistry and Cytochemistry, Vol. 47, 99-112, January 1999, Copyright © 1999, The Histochemical Society, Inc.

ARTICLE

Ultrastructural Localization of ß-Actin and Amphoterin mRNA in Cultured Cells: Application of Tyramide Signal Amplification and Comparison of Detection Methods

Eeva-Liisa Punnonena, Carole Fagesa, Jorma Wartiovaaraa, and Heikki Rauvalaa
a Institute of Biotechnology and Department of Biosciences, University of Helsinki, Helsinki, Finland

Correspondence to: Eeva-Liisa Punnonen, Inst. of Biotechnology, PO Box 56, FIN-00014 University of Helsinki, Finland. E-mail: Eeva-LiisaPunnonen@Helsinki.Fi.

We describe a nonradioactive preembedding in situ hybridization protocol using digoxigenin-labeled RNA probes and tyramide signal amplification to increase the sensitivity of detection. The protocol is sensitive enough for electron microscopic localization of endogenous messenger RNAs encoding ß-actin and amphoterin. Three visualization methods were compared: diaminobenzidine enhanced by nickel, Nanogold enhanced by silver and gold toning, and fluorescently labeled tyramides. Diaminobenzidine and Nanogold can be used in both light and electron microscopy. The nickel-enhanced diaminobenzidine was the most sensitive visualization method. It is easy to accomplish but a drawback is poor spatial resolution, which restricts its use at high magnifications. Nanogold visualization has considerably better spatial resolution and is therefore recommended for electron microscopy. Fluorescent tyramides, especially TRITC–tyramide, offer a good detection method for fluorescence and confocal microscopy. The methods were used to localize amphoterin and ß-actin mRNAs in motile cells. Both mRNAs were found in the soma and cell processes. In double labeling experiments, ß-actin mRNA localized to filamentous structures that also contained ribosomal proteins. Especially in the cortical cytoplasm, ß-actin mRNA was associated with actin filaments. Direct localization to microtubules was only rarely seen. (J Histochem Cytochem 47:99–112, 1999)

Key Words: in situ hybridization, electron microscopy, tyramide signal amplification, mRNA localization, ß-actin, amphoterin


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