DNA的特定染色甲基绿在胚胎的荧光标记的应用

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.净化染料通过将0.4g污点粉末在10ml蒸馏水制备甲基绿的4％水溶液。调匀，直至完全溶解它。 在通风罩，具有至少2份氯仿混合。事先检查管是否氯仿抗性是重要的。 调匀，离心1分钟，在2000 XG加速相分离。 离心后，上层水相用甲基绿，并用氯仿下​​部的有机相和通常的杂质结晶紫被获得。 恢复上相和，直到下阶段似乎完全没有结晶紫重复此步骤。 在?…

Representative Results

厚标本核染色。本文描述的协议允许实现深层结构整体胚胎均匀染色。显影透镜晶核形式48 HPF斑马鱼胚胎中均匀标记（ 图1）。 甲基绿DNA染色允许的细胞周期依赖性的形态差异判别如核分裂或凋亡细胞核（ 图2A）的鉴定。在高分辨率亚核结构也是明显所示为斑马鱼胚胎（ 图2B）的表皮细胞核。 甲基绿是耐在高强度照?…

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

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