The authors would like to acknowledge Patxi Jaso for the production of the video, PEDECIBA and Agencia Nacional de Investigaci&#243;n e Innovaci&#243;n (ANII) for partial funding.

1. A purificação do corante <ol><li> Prepara-se uma solução aquosa a 4% de verde de metilo por dissolução de 0,4 g de pó de mancha em 10 ml de água destilada. Misture bem até dissolver completamente. </li><li> Sob um fume-hood, misturá-lo com pelo menos 2 partes de clorofórmio. É importante verificar com antecedência se os tubos são resistentes ao clorofórmio. </li><li> Misturar bem e centrifuga-se durante 1 min a 2000 xg para acelerar a separação de fases. </li><li> Após centrifugação, a fase aquosa superior com verde de metilo e uma fase orgânica inferior com clorofórmio e o violeta de cristal de contaminantes são obtidos habitual. </li><li> Recuperar a fase superior e repita esta etapa até a fase mais baixa parece completamente desprovida de cristal violeta. </li><li> No final, a fase superior é recuperada e diluído em água até uma concentração final de 2% com verde de metilo. Esta solução de reserva é estável sobre a bancada durante meses e não necessita de ser protegido da luz. </li></ol><ol><li> Fixar os embriões durante a noite em 4% de formaldeído (paraformaldeído) a partir de em tampão fosfato salino (PBS). NOTA: Este pode levar desde algumas horas até durante a noite, dependendo da espessura dos embriões. No exemplo, nós fixa 48 horas pós-fertilização (HPF) embriões de peixe-zebra durante a noite. </li><li> Lavar os embriões três vezes em PBS-T (1% de Triton X-100 em PBS) durante 5 min. A concentração elevada de detergente permeabiliza a cutícula. </li><li> Prepara-se uma solução de 1: 10.000 de verde de metilo (a partir da solução stock 2%) em PBS-T: 5,000-1. A presença de detergente durante a incubação é útil para amostras de espessura, tais como embriões de peixe-zebra e larvas. </li><li> Incubar os embriões na solução, durante pelo menos 6 horas a 4 ° C, com agitação suave. Mais uma vez, a espessura do embrião é o parâmetro de calibração para o comprimento da incubação. </li><li> Lavar os embriões 3 times com PBS durante 30 min para remover o excesso de detergente. Metil fluorescência verde só é evidente quando ligado ao DNA, daí fundo da molécula não ligada é insignificante. </li><li> Montagem em um slide escavado ou de uma câmara caseira com solução de montagem (75% de glicerol, 0,1 M Tris-HCl pH 8). NOTA: Se a coloração nuclear deve ser realizada após imunomarcação, verde metilo pode ser incorporado à solução de montagem na concentração de trabalho, sem a necessidade de lavá-lo afastado. </li><li> Loja refrigerado ou iniciar imagiologia imediatamente. Realizar imagem com um confocal de varredura a laser (ou outra fluorescência) microscópio com excitação a 633 nm e emissão filtros definidos como registrar em 650-750 nm tendo em conta que metil emissão verde atinge o seu máximo em 677 nm. NOTA: Nós fornecemos os dados de medição do perfil de emissão verde de metilo, a ser carregados no software de aquisição, como material suplementar. </li></ol> 3. Fluorescent Nuclear Staining de Whole Pintainho embriões usando Metil Verde <ol><li> Incubar ovos de galinha fertilizados para a fase de interesse. Fixar os embriões durante a noite em 4% de formaldeído (paraformaldeído a partir de) em PBS a 4 ° C. Lavar os embriões 3 vezes em PBS-T, durante 2 h. </li><li> Prepara-se uma solução de 1: 10.000 de verde de metilo (a partir da solução stock 2%) em PBS-T: 5,000-1. Incubar os embriões na solução de coloração durante a noite a 4 ° C. Lavar os embriões 3 vezes em PBS, 1 h cada. Mount na câmara com 75% de glicerol, 0,1 M Tris, pH 8. </li></ol> 4. Criação de Imagens fluorescente etiquetado Núcleos em embriões inteiros montado <ol><li> Preparar uma câmara de imagem furando várias camadas de fita isolante sobre uma lâmina de microscópio e cuidadosamente cortar um buraco para ele com um bisturi para colocar os embriões dentro. Isso impedirá que os embriões de esmagamento durante o exame. </li><li> Transferir cuidadosamente os embriões para a câmara de imagiologia e encher a câmara com a parte superior com 75% de glicerol, Tris-HCl 0,1 M, pH = 8 para umaformação de bolhas vazio. </li><li> Cubra com um # 0 lamela evitar ou eliminar transbordante médio. Selá-lo com unha polonês. </li><li> Aquisição de imagens do espécime no microscópio de fluorescência equipado com um filtro de excitação de 633 ou linha de laser. NOTA: filtros de emissão deve cobrir o máximo de 677 emissões de verde metilo. </li></ol>

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

The authors have nothing to disclose.

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...

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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl green</td>
			<td>Dr. G. Gr&#252;bler</td>
			<td></td>
			<td>Also tested Sigma-Aldrich 323829, which is crystal violet-free</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscopy Leica TCS-SP5</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin&#8211;Tetramethylrhodamine B isothiocyanate</td>
			<td>Sigma-Aldrich</td>
			<td> 
			P1951&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>372978</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R</td>
			<td>eppendorf</td>
			<td>5811 000.010&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>Deltalab</td>
			<td>D100004</td>
			<td></td>
		</tr>
		<tr>
			<td>cover slips</td>
			<td>Esco optics</td>
			<td>R525025</td>
			<td></td>
		</tr>
	</tbody>
</table>

application of the dna-specific stain methyl green in the fluorescent labeling of embryos

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.

A coloração nuclear de amostras grossas. O protocolo aqui descrito permite a realização de coloração homogênea de estruturas profundas no embriões inteiros. Lente desenvolvimento formar núcleos 48 hpf embriões de peixe-zebra são homogeneamente marcado (Figura 1). Coloração de ADN com verde de metilo permite a discriminação do ciclo celular dependentes diferenças morfológicas, tais como a identificação de figuras mitóticas ou núcleos apoptóticos ...

Watch this Scientific Journal Video about Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos at JoVE.com

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

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.

Aplicação do DNA-metil específico Stain Verde no Labeling fluorescentes de Embriões

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

application-dna-specific-stain-methyl-green-fluorescent-labeling

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Developmental Biology

14.0K visualizações.  Institut Pasteur de Montevideo. 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.