Ultrastructural localization of beta-actin and amphoterin mRNA in cultured cells: application of tyramide signal amplification and comparison of detection methods.
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 beta-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 beta-actin mRNAs in motile cells. Both mRNAs were found in the soma and cell processes. In double labeling experiments, beta-actin mRNA localized to filamentous structures that also contained ribosomal proteins. Especially in the cortical cytoplasm, beta-actin mRNA was associated with actin filaments. Direct localization to microtubules was only rarely seen. (J Histochem Cytochem 47:99-112, 1999)
Punnonen
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EL
Institute of Biotechnology and Department of Biosciences, University of Helsinki, Helsinki, Finland. Eeva-Liisa.Punnonen@Helsinki.Fi
Fages
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Wartiovaara
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Rauvala
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D000199:Actins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D014408:Biomarkers, Tumor / Q000032:analysis; D001710:Biotin / Q000031:analogs & derivatives* / Q000032:analysis; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics*; D004396:Coloring Agents; D003599:Cytoskeleton / Q000737:chemistry; D024243:HMGB1 Protein; D006609:High Mobility Group Proteins / Q000032:analysis / Q000235:genetics*; D017403:In Situ Hybridization / Q000379:methods*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D011554:Pseudopodia / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D014439:Tyramine / Q000031:analogs & derivatives* / Q000032:analysis; D004124:p-Dimethylaminoazobenzene / Q000032:analysis
D000199:Actins; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D004396:Coloring Agents; D024243:HMGB1 Protein; D006609:High Mobility Group Proteins; D012333:RNA, Messenger; C111551:biotinyltyramide; D001710:Biotin; D004124:p-Dimethylaminoazobenzene; D014439:Tyramine