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.

Boyar 1. Saflaştırma <ol><li> Damıtılmış su, 10 ml leke tozu 0.4 g çözülmesiyle metil yeşili bir% 4 sulu çözelti hazırlayın. Tamamen eritilmesi gelene kadar iyice karıştırınız. </li><li> Bir duman-Kaputun altında, kloroform en az 2 parçaları ile karıştırarak. Bu tüpler kloroform dayanıklı olup olmadığını önceden kontrol edilmesi önemlidir. </li><li> İyice karıştırın ve faz ayrımını hızlandırmak için 2,000 xg&#39;de 1 dakika boyunca santrifüj. </li><li> Santrifüj işleminden sonra, metil yeşili bir üst sulu faz ve kloroform ile daha düşük bir organik faz ve her zamanki gibi kirletici kristal viyole elde edilir. </li><li> Üst fazı kurtarmak ve alt faz kristal viyole tamamen yoksun görünene kadar bu adımı tekrarlayın. </li><li> Sonunda, üst faz elde edilir ve% 2&#39;lik bir metil yeşili bir nihai konsantrasyona kadar su ile seyreltilir. Bu stok çözeltisi, ay tezgahında stabil olduğu ve ışıktan korunması gerekmez. </li></ol><ol><li> Fosfat tamponlu tuzlu su (PBS) içinde (paraformaldehid ile ilgili) bir karışımı,% 4 formaldehid embriyolar sabitleyin. NOT: Bu bir kaç saat sürebilir gecede embriyoların kalınlığına bağlı olarak. Örneğin, biz bir gecede 48 saat sonrası fertilizasyon (hpf) zebrafish embriyolar sabit. </li><li> 5 dakika boyunca PBS-T (PBS içinde% 1 Triton X-100), embriyolar üç kez yıkayın. deterjanın yüksek konsantrasyonu, kütikül permeabilizes. </li><li> 5,000-1: PBS-T içinde (% 2 stok çözeltiden) metil yeşili 10,000 çözeltisi 1 hazırlayın. inkübasyon sırasında deterjan varlığının örneğin zebra balığı embriyolarının ve larva gibi kalın örneklerde, için kullanışlıdır. </li><li> Hafifçe çalkalanarak, 4 ° C de en az 6 saat boyunca çözelti içinde embriyolar inkübe edin. Yine, embriyo kalınlığı inkübasyon süresi için kalibrasyon parametredir. </li><li> Embriyoların 3 tim yıkayın30 dakika boyunca PBS ile ES deterjan fazlasının ayrılması için. DNA&#39;ya bağlandığı zaman metil, yeşil floresan özelliğinin yalnızca belirgindir, bağlanmamış molekülden dolayısıyla arka ihmal edilebilir düzeydedir. </li><li> Bir kazı slayt veya montaj çözeltisi ile yapımı bölmesi üzerine monte (% 75 gliserol, 0.1 M Tris-HCI, pH 8). NOT: Nükleer boyanma immunolabeling sonra yapılacak ise, metil yeşil bunu yıkayacak gerek kalmadan, çalışma konsantrasyonunda montaj çözüm eklenebilir. </li><li> Mağaza buzdolabında ya da hemen görüntüleme başlatın. 650-750 nm yeşil emisyon 677 nm maksimum ulaşır metil dikkate alarak kayıt ayarlanmış 633 nm ve emisyon filtreleri de uyarma ile bir lazer tarama konfokal (ya da diğer floresan) mikroskop ile görüntüleme gerçekleştirin. NOT: metil yeşil emisyon profili ölçülen verileri, ek malzeme olarak, satın alma yazılımı yüklenecek sağlamak. </li></ol> 3. Floresan Nükleer StMetil Yeşil kullanarak Tüm Chick Embriyolar Tahliye <ol><li> Ilgi sahneye döllenmiş tavuk yumurtaları kuluçkaya yatmaktadır. 4 ° C&#39;de bir gece boyunca PBS (paraformaldehid ile ilgili)% 4 formaldehit embriyolar düzeltildi. 2 saat süre ile, PBS-T içinde embriyolar 3 kere yıkayın. </li><li> 5,000-1: PBS-T içinde (% 2 stok çözeltiden) metil yeşili 10,000 çözeltisi 1 hazırlayın. Gece boyunca 4 ° C sıcaklıkta boyama çözeltisi embriyolar inkübe edin. Embriyolar 3 kere PBS ile 1 saat, her yıkayın. % 75 gliserol, 0.1 M Tris, pH 8, oda içinde monte edin. </li></ol> 4. Görüntüleme Floresan Tüm monte Embriyolar Çekirdeği Etiketli <ol><li> Dikkatli bir mikroskop lamı üzerine elektrik bandı birkaç kat yapışmasını tarafından bir görüntüleme odası hazırlayın içinde embriyolar yerleştirmek için bir neşter ile içine bir delik kesti. Bu görüntüleme sırasında ezilme embriyolar önleyecektir. </li><li> Dikkatli bir şekilde% 75 gliserol, 0.1 M Tris-HCI, pH = 8, üstüne bölmeyi görüntüleme odasına embriyo transferi ve dolgugeçersiz kabarcık oluşumu. </li><li> Bir # 0 lamel kaçınarak veya orta taşan ortadan ile kaplayın. Oje ile mühür. </li><li> Bir 633 uyarılma filtreli veya lazer hattı ile teçhiz edilmiş bir flüoresans mikroskobu, numunenin görüntüleri elde edin. NOT: Emisyon filtreleri metil yeşili 677 emisyon maksimum kapsamalıdır. </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.

Kalın numunelerin Nükleer boyanma. Burada tarif edilen protokol bütün embriyolar derin yapıların homojen lekelenme başarı sağlar. Gelişmekte mercek çekirdek formu 48 hpf Zebra balığı embriyolar homojen (Şekil 1) etiketlenir. Metil yeşil DNA boyama gibi mitoz veya apoptotik çekirdeklerin (Şekil 2A) tanımlanması gibi hücre döngüsü bağımlı morfolojik farklılıkların ayrımcılığa izin verir. Zebra balığı embriyolar (Şe...

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.

Embriyolar Floresan Etiketleme DNA Özgü Leke Metil Green Uygulaması

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 Görüntüleme.  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.