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Actin Staining Techniques
The successful imaging of cytoskeletal organization and dynamics can be achieved by the visualization of the actin cytoskeleton in cells or tissues using fluorescent markers.
In contrast to the current approaches, LifeAct visualizes F-actin within fixed and living cells, without compromising cellular processes, such as the locomotion of dendritic cells or neuronal polarization. More >>
Live cell imaging has become a highly important microscopy method, because genetically encoded fluorophores (e.g., green fluorescent protein (GFP)) have become suitable for use in eukaryotic cells. Researchers are able to instantly visualize the localization and dynamics of one of these fusion proteins by coupling fluorescent proteins (FPs) to endogenous proteins. Actin, coupled to FPs, and also actin-binding proteins, are readily used for the visualization of the cytoskeleton. However, these widely used markers have certain limitations of use, such as, when fusion proteins show reduced functionality, as compared to the endogenous ones, and therefore alter the actin dynamics. This is also true with other markers. The novel actin marker LifeAct overcomes these limitations. It does not interfere with cytoskeletal dynamics and leads to unrestricted actin dynamics.
Nowadays, with usage of high-end microscopy tools (e.g., TIRF or STED), it is becoming even more important to be able to generate reliable and authentic data from live cell imaging. This applies also to the markers that are used for labeling the proteins in cells.
Before live cell imaging was available, like it is today, the only marker for F-actin was Phalloidin coupled to different fluorophores (e.g., rhodamine). Phalloidin originates from the Amanita phalloides mushroom. It is toxic because it irreversibly binds together filamentous actin, which paralyzes the cell’s cytoskeleton and leads to cell (or organism) death. Obviously, this is a disadvantage when working with living cells, and any generated data needs to be treated carefully. Phalloidin, therefore, is recommended for use only with fixed samples.
In contrast to Phalloidin, the novel actin marker LifeAct visualizes F-actin within fixed and living cells, without disturbing actin kinetics and functionality.