The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.
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The authors would like to thank Dr. Cristina Arruti and Dr. Pablo Oppezzo for facilitating the use of reagents and lab installations; the Cell Biology Unit at the Institute Pasteur Montevideo for access to the confocal microscope and flow cytometer; Dr. Mario Señorale for access to the G:Box. Partial funding was from Comisión Sectorial de Investigación Científica (CSIC)-Universidad de la República and PEDECIBA, Uruguay.
Electronic supplementary material
Online Resource 1 (Movie)3D reconstruction of a mitotic cell from chick embryo stained with MG, Hoechst 33342 and TRITC-Phalloidin showing that MG provides very good levels of image resolution at x/y and z axes. Green, MG; blue, Hoechst 33342 (shown merged with the green channel); red, TRITC-Phalloid (mpg 1716 kb)
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Prieto, D., Aparicio, G., Morande, P.E. et al. A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green. Histochem Cell Biol 142, 335–345 (2014). https://doi.org/10.1007/s00418-014-1215-0
- Methyl green
- Flow cytometry
- Confocal microscopy