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Genetic encoded multilabeling is essential for modern cell biology. In fluorescence microscopy this need has been satisfied by the development of numerous color-variants of the green fluorescent protein. In electron microscopy, however, true genetic encoded multilabeling is currently not possible. Here, we introduce combinatorial cell organelle type-specific labeling as a strategy for multilabeling. First, we created a reliable and high sensitive label by evolving the catalytic activity of horseradish peroxidase (HRP). We then built fusion proteins that targeted our new enhanced HRP (eHRP) to three cell organelles whose labeling pattern did not overlap with each other. The labeling of the endoplasmic reticulum, synaptic vesicles and the plasma membrane consequently allowed for triple labeling in the EM. The combinatorial expression of the three organelle-specific constructs increased the number of clearly distinguishable labels to seven. This strategy of multilabeling for EM closes a significant gap in our tool set and has a broad application range in cell biology.

Quintuple labeling in the electron microscope with genetically encoded enhanced horseradish peroxidase