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

Abstract

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 β-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 β-actin mRNAs in motile cells. Both mRNAs were found in the soma and cell processes. In double labeling experiments, β-actin mRNA localized to filamentous structures that also contained ribosomal proteins. Especially in the cortical cytoplasm, β-actin mRNA was associated with actin filaments. Direct localization to microtubules was only rarely seen.