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. Purificazione del colorante <ol><li> Preparare una soluzione acquosa al 4% del verde metile sciogliendo 0,4 g di colorante in polvere in 10 ml di acqua distillata. Mescolare accuratamente fino a completa dissoluzione. </li><li> Sotto una cappa-cappa, mescolare con almeno 2 parti di cloroformio. E &#39;importante verificare in anticipo se i tubi sono resistenti cloroformio. </li><li> Mescolare accuratamente e centrifugare per 1 minuto a 2000 xg per accelerare la separazione di fase. </li><li> Dopo centrifugazione, una fase superiore acquosa con verde metile e una fase organica inferiore con cloroformio e la solita contaminante cristalvioletto sono ottenuti. </li><li> Recuperare la fase superiore e ripetere l&#39;operazione fino a quando appare fase inferiore del tutto privo di cristallo viola. </li><li> Al termine, la fase superiore viene recuperato e diluito in acqua ad una concentrazione finale del 2% verde metile. Questa soluzione madre è stabile sul banco di lavoro per mesi e non deve essere protetto dalla luce. </li></ol><ol><li> Fissare gli embrioni durante la notte nel 4% di formaldeide (da paraformaldeide) in tampone fosfato (PBS). NOTA: Questo può richiedere da alcune ore a seconda overnight dello spessore degli embrioni. Nell&#39;esempio, abbiamo fissato 48 hr post-fecondazione (HPF) zebrafish embrioni durante la notte. </li><li> Lavare gli embrioni tre volte in PBS-T (1% Triton X-100 in PBS) per 5 min. L&#39;elevata concentrazione di detersivo permeabilizes cuticola. </li><li> Preparare un 1: 5,000-1: 10.000 soluzione di verde metile (dal 2% soluzione madre) in PBS-T. La presenza di detersivo durante l&#39;incubazione è utile per i campioni spessi, come zebrafish embrioni e larve. </li><li> Incubare gli embrioni nella soluzione per almeno 6 ore a 4 ° C, con dolce dondolio. Anche in questo caso, lo spessore dell&#39;embrione è il parametro di calibrazione per la lunghezza dell&#39;incubazione. </li><li> Lavare gli embrioni 3 times con PBS per 30 minuti per eliminare l&#39;eccesso di detersivo. Metile fluorescenza verde è solo evidente quando si lega al DNA, quindi sfondo dalla molecola non legato è trascurabile. </li><li> Montare su un vetrino scavato o una camera in casa con soluzione di montaggio (75% glicerolo, 0,1 M Tris · HCl pH 8). NOTA: Se la colorazione nucleare deve essere eseguita dopo immunomarcatura, verde metile può essere incorporato alla soluzione di montaggio alla concentrazione di lavoro, senza la necessità di lavare via. </li><li> Conservare in frigorifero o avviare immediatamente l&#39;imaging. Eseguire l&#39;imaging con un confocale a scansione laser (o altro a fluorescenza) microscopio con eccitazione a 633 nm ed emissione filtri impostati per registrare a 650-750 nm, tenendo conto del fatto che le emissioni di metile verde raggiunge il suo massimo a 677 nm. NOTA: Forniamo i dati misurati del profilo emissioni verde metile, per essere caricati nel software di acquisizione, come materiale supplementare. </li></ol> 3. fluorescente nucleare StAining di Whole Chick embrioni utilizzando metile Verde <ol><li> Incubare le uova di gallina fecondate allo stadio di interessi. Fissare gli embrioni notte in formaldeide al 4% (da paraformaldeide) in PBS a 4 ° C. Lavare gli embrioni 3 volte in PBS-T per 2 ore. </li><li> Preparare un 1: 5,000-1: 10.000 soluzione di verde metile (dal 2% soluzione madre) in PBS-T. Incubare gli embrioni nella soluzione colorante notte a 4 ° C. Lavare gli embrioni 3 volte in PBS, 1 ora ciascuno. Montare in camera con il 75% di glicerolo, 0,1 M Tris, pH 8. </li></ol> 4. Imaging fluorescente Nuclei in embrioni interi montato <ol><li> Preparare una camera di imaging incollando diversi strati di nastro isolante su un vetrino da microscopio e accuratamente tagliato un foro in esso con un bisturi per posizionare gli embrioni all&#39;interno. Questo consentirà di evitare gli embrioni da schiacciamento durante l&#39;imaging. </li><li> Trasferire accuratamente gli embrioni alla camera di imaging e riempire la camera verso l&#39;alto con il 75% glicerolo, 0,1 M Tris-HCl, pH = 8 con unla formazione di bolle vuoto. </li><li> Coprire con un # 0 vetrino evitare o eliminare media straripante. Sigillare con smalto. </li><li> Acquisire immagini del campione su un microscopio a fluorescenza dotato di un filtro di eccitazione 633 o linea laser. NOTA: i filtri delle emissioni dovrebbero riguardare al massimo 677 emissione di verde metile. </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.

Colorazione nucleare dei campioni di spessore. Il protocollo qui descritto consente il conseguimento di colorazione omogenea delle strutture profonde in embrioni interi. Lenti in via di sviluppo formare nuclei 48 hpf embrioni di zebrafish sono omogeneamente etichettati (Figura 1). Metile colorazione verde DNA permette la discriminazione di ciclo cellulare differenze morfologiche a carico, come l&#39;individuazione di figure mitotiche o nuclei apoptotici (Figura 2A).<...

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.

Applicazione del DNA-Specific Stain metile Verde al Fluorescent Etichettatura degli embrioni

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

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14.0K Views.  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.