Summary

Применение ДНК-специфичных красителей метиловым зеленым в Флуоресцентная маркировка эмбрионов

Published: May 02, 2015
doi:

Summary

A method for fluorescent staining of fixed biological material with the specific DNA label methyl green is described. Methyl green is used in a diluted aqueous solution and is very resistant to photobleaching. Its far-red emission allows for deep specimen imaging, making it particularly adequate for whole embryos.

Abstract

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.

Introduction

Fluorescent staining of DNA is broadly and routinely used both in biological research and in clinical diagnosis, either for the detailed study of nuclear and chromosomal structure, for counterstaining tissues and cells, or for studying cell cycle parameters1. Although several DNA stains are available, the most widely used have been those excited by UV light and emitting in blue, which fit in the usual three-filter systems used in most conventional epifluorescence microscopes when combined with green and orange-red-emitting fluorophores, such as fluorescent proteins or small molecules attached to antibodies. Among these blue-emitting DNA stains, the most common are DAPI and Hoechst minor groove-binding agents2,3. Nonetheless, the incorporation of a wide variety of laser emission lines and spectral detection in laser scanning microscopes, has allowed researchers to choose for the use of far-red-emitting nuclear stains such as propidium iodide (PI) or TO-PRO 34. An advantage of these stains is that they overcome the problem of autofluorescence found in many cells and tissues, particularly in embryos5, but many of them have different problems such as having wide emission profiles or being very sensitive to photobleaching6.

The Swiss researcher Friedrich Miescher discovered DNA in 1869, extracting it from the nuclei of white blood cells found in pus. He called it nuclein, and showed a few years later that it formed precipitates with the relatively new stain methyl green. Methyl green is composed by three aniline rings, with different degrees of methylation, and has two positive charges that allow it to strongly bind to the major groove of DNA7,8. DNA staining by methyl green was very early shown to be specific, leading to the development by Unna and Pappenheim of a combined stain with pyronin, which specifically labels RNA. Methyl green-pyronin has since then been extensively used in routine histological technique9.

Until recently, very little was known about the fluorescent properties of methyl green. Our previous work, however, has uncovered some of methyl green spectral advantages6 such as its excitation at 633 nm, a convenient Stokes shift, its emission on the near-infrared portion of the electromagnetic spectrum, higher photostability than commercially available DNA stains with similar spectral properties and at a very low cost. This is why we decided to test it for being used as a very high affinity and specific fluorescent label for DNA on embryonic tissues and in whole embryos. Furthermore, until now methyl green nuclear staining was performed at low pH, making it difficult to combine with antibody staining. The goal of the method detailed here is to have a protocol for fluorescent nuclear DNA staining which overcomes the aforementioned inconvenience by performing the staining procedure at physiological pH, thus making it a suitable counterstain for immunolabeling on sections and whole tissues.

Protocol

1. Очистка красителя Подготовка 4% -ный водный раствор метиловым зеленым при растворении 0,4 г порошка красителя в 10 мл дистиллированной воды. Смешивайте до полного его растворения. Под вытяжного шкафа, смешать его с, по крайней мере 2-х частей хлороформа. Важно заранее проверит?…

Representative Results

Ядерная окрашивание толстых образцах. Протокол описано позволяет для достижения однородной окраски глубинных структур в целом эмбрионов. Развивающиеся объектив ядра образуют 48 эмбрионы рыбок данио HPF однородно помечены (рис 1). Метил зеленый окрашивание ДНК п?…

Discussion

The progressive development of microscopy techniques in the last decades, with a particular focus in variations of fluorescent microscopy, have led to the possibility of exploring the dynamics and structure of intact tissue samples, including whole embryos10,11. One major limitation has been, however, the availability of good quality fluorophores that can be used to directly and quickly stain subcellular structures.

The most frequently used fluorescent DNA stains, such as DAPI or H…

Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors would like to acknowledge Patxi Jaso for the production of the video, PEDECIBA and Agencia Nacional de Investigación e Innovación (ANII) for partial funding.

Materials

Glycerol Sigma-Aldrich G5516
Methyl green Dr. G. Grübler Also tested Sigma-Aldrich 323829, which is crystal violet-free
Paraformaldehyde Sigma-Aldrich 158127
Laser scanning confocal microscopy Leica TCS-SP5 Leica Microsystems
Phalloidin–Tetramethylrhodamine B isothiocyanate Sigma-Aldrich
P1951 
chloroform Sigma-Aldrich 372978
Centrifuge 5810 R eppendorf 5811 000.010  
microscope slides Deltalab D100004
cover slips Esco optics R525025

References

  1. Klonisch, T., Wark, L., Hombach-Klonisch, S., Mai, S. Nuclear imaging in three dimensions: a unique tool in cancer research. Annals of anatomy. 192 (5), 292-301 (2010).
  2. Kapuscinski, J. DAPI: a DNA-specific fluorescent probe. Biotechnic. 70 (5), 220-233 (1995).
  3. Latt, S. A., Stetten, G. Spectral studies on 33258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis. The journal of histochemistry and cytochemistry. 24 (1), 24-33 (1976).
  4. Van Hooijdonk, C. A., Glade, C. P., Van Erp, P. E. TO-PRO-3 iodide: a novel HeNe laser-excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry. Cytometry. 17 (2), 185-189 (1994).
  5. Beumer, T. L., Veenstra, G. J., Hage, W. J., Destrée, O. H. Whole-mount immunohistochemistry on Xenopus embryos using far-red fluorescent dyes. Trends in genetics. 11 (1), 9 (1995).
  6. Prieto, D., Aparicio, G., Morande, P. E., Zolessi, F. R. A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green. Histochemistry and cell biology. 142 (3), 335-345 (2014).
  7. Kim, S. K., Nordén, B. Methyl green. A DNA major-groove binding drug. FEBS letters. 315 (1), 61-64 (1993).
  8. Nordén, B., Tjerneld, F. Binding of methyl green to deoxyribonucleic acid analyzed by linear dichroism. Chemical Physics Letters. 50 (3), 508-512 (1977).
  9. Lyon, H., Jakobsen, P., Andersen, a. P. Standardized methyl green-pyronin Y procedures using pure dyes. The Histochemical journal. 18 (2-3), 90-94 (1986).
  10. Amat, F., Keller, P. J. Towards comprehensive cell lineage reconstructions in complex organisms using light-sheet microscopy. Development, growth & differentiation. 55 (4), 563-578 (2013).
  11. Chung, K., Deisseroth, K. CLARITY for mapping the nervous system. Nature methods. 10 (6), 508-5013 (2013).
  12. Lenz, P. Fluorescence measurement in thick tissue layers by linear or nonlinear long-wavelength excitation. Applied. 38 (16), 3662-3669 (1999).

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Cite This Article
Prieto, D., Aparicio, G., Machado, M., Zolessi, F. R. Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos. J. Vis. Exp. (99), e52769, doi:10.3791/52769 (2015).

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