Back to Search
Start Over
Quintuple labeling in the electron microscope with genetically encoded enhanced horseradish peroxidase.
- Source :
-
PloS one [PLoS One] 2018 Jul 16; Vol. 13 (7), pp. e0200693. Date of Electronic Publication: 2018 Jul 16 (Print Publication: 2018). - Publication Year :
- 2018
-
Abstract
- 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.<br />Competing Interests: The authors have declared that no competing interests exist.
- Subjects :
- Animals
Cell Line
Horseradish Peroxidase biosynthesis
Horseradish Peroxidase genetics
Humans
Mice
Cell Membrane genetics
Cell Membrane metabolism
Cell Membrane ultrastructure
Endoplasmic Reticulum genetics
Endoplasmic Reticulum metabolism
Endoplasmic Reticulum ultrastructure
Microscopy, Electron
Staining and Labeling methods
Synaptic Vesicles genetics
Synaptic Vesicles metabolism
Synaptic Vesicles ultrastructure
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 13
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 30011315
- Full Text :
- https://doi.org/10.1371/journal.pone.0200693