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 common are DAPI and Hoechst minor groove-binding agents^2,3. 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 DNA^7,8. 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. 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 until lower phase appears completely devoid of crystal violet.
6. At the end, the upper phase is recovered and diluted in water to a final concentration of 2% methyl green. This stock solution is stable on the workbench for months and does not need to be protected from light.

2. Fluorescent Nuclear Staining of Whole Zebrafish Embryos using Methyl Green

1. Fix the embryos overnight in 4% formaldehyde (from paraformaldehyde) in phosphate buffered saline (PBS).
   NOTE: This can take from a few hours to overnight depending on the thickness of the embryos. In the example, we fixed 48 hr post-fertilization (hpf) zebrafish embryos overnight.
2. Wash the embryos three times in PBS-T (1% Triton X-100 in PBS) for 5 min. The elevated concentration of detergent permeabilizes the cuticle.
3. Prepare a 1:5,000-1:10,000 solution of methyl green (from 2% stock solution) in PBS-T. The presence of detergent during the incubation is useful for thick specimens, such as zebrafish embryos and larvae.
4. Incubate the embryos in the solution for at least 6 hr at 4 °C, with gentle rocking. Again, the thickness of the embryo is the calibrating parameter for the length of the incubation.
5. Wash the embryos 3 times with PBS for 30 min to remove the detergent excess. Methyl green fluorescence is only evident when bound to DNA, hence background from the unbound molecule is negligible.
6. Mount on an excavated slide or a homemade chamber with mounting solution (75% glycerol, 0.1 M Tris·HCl pH 8).
   NOTE: If the nuclear staining is to be performed after immunolabeling, methyl green can be incorporated to the mounting solution at the working concentration, with no need to wash it away.
7. Store refrigerated or start imaging immediately. Perform imaging with a laser scanning confocal (or other fluorescence) microscope with excitation at 633 nm and emission filters set to register at 650-750 nm taking into account that methyl green emission reaches its maximum at 677 nm.
   NOTE: We provide the measured data of the methyl green emission profile, to be loaded into the acquisition software, as supplementary material.

3. Fluorescent Nuclear Staining of Whole Chick Embryos using Methyl Green

1. Incubate fertilized hen eggs to the stage of interest. Fix the embryos overnight in 4% formaldehyde (from paraformaldehyde) in PBS at 4 °C. Wash embryos 3 times in PBS-T for 2 hr.
2. Prepare a 1:5,000-1:10,000 solution of methyl green (from 2% stock solution) in PBS-T. Incubate the embryos in the staining solution overnight at 4 °C. Wash the embryos 3 times in PBS, 1 hr each. Mount in chamber with 75% glycerol, 0.1 M Tris, pH 8.

4. Imaging Fluorescently Labeled Nuclei in Whole-mounted Embryos

1. Prepare an imaging chamber by sticking several layers of electrical tape over a microscope slide and carefully cut a hole into it with a scalpel to place the embryos within. This will prevent embryos from crushing during imaging.
2. Carefully transfer the embryos to the imaging chamber and fill the chamber to the top with 75% glycerol, 0.1 M Tris-HCl, pH=8 to avoid bubble formation.
3. Cover with a #0 coverslip avoiding or eliminating medium overflowing. Seal it with nail polish.
4. Acquire images of the specimen on a fluorescence microscope equipped with a 633 excitation filter or laser line.
   NOTE: Emission filters should cover the 677 emission maximum of methyl green.

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