Nuclear staining is essential for many flow cytometry and fluorescence microscopy experiments. Whether demonstrating nuclear localization of an antigen, counting cells or characterizing general architecture of a tissue, it is extremely helpful to stain the nucleus using a fluorescent probe for double-stranded nucleic acids.
The Molecular Probes TO-PRO 3 Iodide belongs to a family of monomeric, cyanine nucleic acid stains that allow very sensitive DNA detection due to good fluorescence signal intensity. This family of products is used for a wide range of applications, including chromosome counterstaining and nucleic acid staining during gel electrophoresis. In addition, the fluorescence spectra of the Molecular Probes monomeric cyanine nucleic acid stains cover the entire visible wavelenght range. In other words, there is one suited for any need.
Our specific need was to find an alternative to UV-excited, fluorescent probes, such as bis-benzimide (Hoechst) or DAPI. In fact, our confocal microscope is not equipped with the expensive UV (Mercury) source and has only standard visible lasers (488, 543 andf 633 nm). Due to our limited light options, we cannot excite DAPI and visualize the nuclei through standard blue fluorescence. We decided to try the Molecular Probes TO-PRO 3 Iodide for nuclear staining of our samples obatined from either cell cultures or tissue cryosections. This compound was efficiently excited by our laser and emitted fluorescence in the far-red region of the spectrum, enabling triple staining with two additional fluorochromes (e.g. Alexa 488 and Alexa 568). We were happy to verify that a quick (a few minutes incubation) staining of our samples with TO-PRO 3 resulted in a bright nuclear staining that did not interfere with additional, green and red, fluorescent stainings. Other groups in the Department tryed TO-PRO 3 ever since and they also seem happy.
The product is much more expensive than Hoechst or DAPI, and this probably represents the only limitation to a wide use of TO-PRO 3 in our laboraotry. However, it was a pleasant surprise to discover that we could dilute the product up to 10,000 times to make a working solution (10x greater than suggested by the manufacutrer’s instructions) and still have a detectable signal in the confocal microscopy. For this reason the amount of product used for each experiment is very small (thanks to the high concentration of TO-PRO 3 stock) making the cost negligible with respect to that of immunofluorescence. An other pleasant discovery was that the product is quite stable during both prolonged excitation (needed for confocal imaging) and sample storage at –20oC, a useful feature when you want to have a second look to your samples. In conclusion, I would say that the high cost is justified by the very little amount of product needed and by its great technical features.
Molecular Probes TO-PRO 3 Iodide is a novel product for DNA (e.g nuclear) labeling in fluorescence-based applications. It is sensitive, stable and has the interesting property of being excited by visible light. Based on these features, I would recommended it to researchers doing flow cytomentry or confocal microscopy work.
Dario Coletti, PhD
Research Associate
University of Rome La Spaienza