Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

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 com....

Protocol

1. Purification of the Dye

1. Prepare a 4% aqueous solution of methyl green by dissolving 0.4 g of stain powder in 10 ml distilled water. Mix thoroughly until completely dissolving it.
2. Under a fume-hood, mix it with at least 2 parts of chloroform. It is important to check beforehand whether the tubes are chloroform resistant.
3. Mix thoroughly and centrifuge for 1 min at 2,000 x g to accelerate phase separation.
4. After centrifugation, an upper aqueous phase with methyl green and a lower organic phase with chloroform and the usual contaminant crystal violet are obtained.
5. Recover the upper phase and repeat this step unti....

Results

Nuclear staining of thick specimens. The protocol herein described allows for the achievement of homogeneous staining of deep structures in whole embryos. Developing lens nuclei form 48 hpf zebrafish embryos are homogeneously labeled (Figure 1).

Methyl green DNA staining allows discrimination of cell cycle dependent morphological differences such as the identification of mitotic figures or apoptotic nuclei (Figure 2A). Subnuclear structures at high resolution .......

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 embryos^10,11. 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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