Abstract
For nearly 50 years immunogold labeling on ultrathin sections has been successfully used for protein localization in laboratories worldwide. In theory and in practice, this method has undergone continual improvement over time. In this study, we carefully analyzed circulating protocols for postembedding labeling to find out if they are still valid under modern laboratory conditions, and in addition, we tested unconventional protocols. For this, we investigated immunolabeling of Epon-embedded cells, immunolabeling of cells treated with osmium, and the binding behavior of differently sized gold particles. Here we show that (in contrast to widespread belief) immunolabeling of Epon-embedded cells and of cells treated with osmium tetroxide is actually working. Furthermore, we established a "speed protocol" for immunolabeling by reducing antibody incubation times. Finally, we present our results on three-dimensional immunogold labeling.
Highlights
Around 1890, Emil von Behring and Shibasaburo Kitasato discovered a “specific antitoxic activity” in serum of animals infected with diphtheria
We show that immunolabeling of Epon-embedded cells and of cells treated with osmium tetroxide is working
Immunocytochemical investigations were performed on ultrathin sections of organisms we routinely grow and handle in our laboratories: Ignicoccus hospitalis (I. hospitalis), a hyperthermophilic prokaryote and Phaeodactylum tricornutum (P. tricornutum), a photosynthetic unicellular and eukaryotic microalga
Summary
Around 1890, Emil von Behring and Shibasaburo Kitasato discovered a “specific antitoxic activity” in serum of animals infected with diphtheria. Via auto metallographic processes like silver enhancement, these nano-sized gold particles can be enlarged until the desired size is reached (Danscher, 1981; Stierhof, Hermann, Humbel, & Schwarz, 1995) Another instrument for antigen detection is protein A or G. Due to the strong cross-linking properties of epoxy resins, including reactions with the biological sample, antigenicity of the sample can be reduced (Stirling, 1990) This loss of antigenicity can be lowered when methacrylate resins like Lowicryl or London resins are used instead, because methacrylates can retain biochemical reactivity especially within partial dehydration. We developed an accelerated protocol for protein localization and we compared labeling efficiency of secondary antibodies coupled to gold particles of different sizes. Immunocytochemical investigations were performed on ultrathin sections of organisms we routinely grow and handle in our laboratories: Ignicoccus hospitalis (I. hospitalis), a hyperthermophilic prokaryote and Phaeodactylum tricornutum (P. tricornutum), a photosynthetic unicellular and eukaryotic microalga
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