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.

色素の1精製<ol><li> 10mlの蒸留水で汚れ粉末0.4グラムを溶解することにより、メチルグリーンの4％水溶液を調製します。それを完全に溶解するまで十分に混合します。 </li><li>ヒュームフードの下で、クロロホルムの少なくとも2部とそれを混ぜます。これは、チューブがクロロホルム耐性があるかどうかを事前に確認することが重要です。 </li><li>十分に混合し、相分離を促進するために2,000×gで1分間遠心します。 </li><li>遠心分離後、メチルグリーンと上部の水相、クロロホルム、通常汚染物質クリスタルバイオレットと下層の有機相が得られます。 </li><li>上相を回収し、下相は、クリスタルバイオレットを完全に欠い表示されるまで、この手順を繰り返します。 </li><li>終了時に、上相を回収し、2％のメチルグリーンの最終濃度まで水で希釈しました。このストック溶液を、ヶ月間作業台の上に安定であり、光から保護される必要がありません。 </li></ol><ol><li>リン酸緩衝生理食塩水（PBS）中の（パラホルムアルデヒドから）4％ホルムアルデヒド中で一晩胚を修正しました。 注：これは一晩胚の厚さに応じてするのに数時間かかることがあります。例では、一晩の48時間後に受精（HPF）ゼブラフィッシュの胚を固定しました。 </li><li> 5分間、PBS-T（PBS中1％のTriton X-100）中で胚を3回洗浄します。界面活性剤の高濃度は、キューティクルを透過性に。 </li><li> 5,000-1：PBS-T（2％ストック溶液から）メチルグリーン10,000溶液1を調製します。インキュベーション中の界面活性剤の存在は、ゼブラフィッシュの胚および幼生のような厚い標本のために有用です。 </li><li>緩やかに揺り動かしながら、4℃で少なくとも6時間溶液中で胚を培養します。ここでも、胚の厚さは、培養時間の長さのための校正パラメータです。 </li><li>胚3ティムを洗います洗剤過剰を取り除くために30分間、PBSでES。 DNAに結合した場合にメチルグリーン蛍光は、したがって、非結合分子からのバックグラウンドは無視できる、唯一のは明白です。 </li><li>出土したスライドや実装溶液と自家製室（75％グリセロール、0.1Mトリス·塩酸pHは8）にマウント。 注：核染色は免疫標識後に行われる場合、メチルグリーン、それを洗い流す必要なしに、使用濃度に取り付け溶液に組み込むことができます。 </li><li>ストアは、冷蔵またはすぐに撮影を開始します。レーザー走査共焦点（または他の蛍光）メチルグリーン発光は677 nmで最大となることを考慮して650〜750 nmに登録するように設定さ633 nmおよび発光フィルターで励起し顕微鏡で撮影を行います。 注：私たちは、補足資料として、取得ソフトウェアにロードされるメチルグリーン発光プロファイルの測定データを、提供します。 </li></ol> 3.蛍光原子力セントメチルグリーンを用いた全ニワトリ胚のaining <ol><li>興味のあるステージに受精鶏卵をインキュベートします。 4℃で一晩、PBS中（パラホルムアルデヒドから）4％ホルムアルデヒド中で胚を修正しました。 2時間の胚をPBS-Tで3回洗浄します。 </li><li> 5,000-1：PBS-T（2％ストック溶液から）メチルグリーン10,000溶液1を調製します。 4℃で一晩染色液中の胚を培養します。胚をPBSで3回、1時間ごとに洗浄します。 75％グリセロール、0.1Mトリス、pHが8と、チャンバ内のマウント。 </li></ol> 4.イメージング蛍光が全体に取り付けられた胚中の核を標識<ol><li>顕微鏡スライド上に電気テープのいくつかの層を貼り合わせて画像化チャンバを準備し、慎重内の胚を配置するメスでそれに穴をカット。これは、撮像中に破砕から胚を防ぐことができます。 </li><li>慎重に75％グリセロール、0.1 Mトリス-HCl、pH値= 8でトップにチャンバーを撮像チャンバーに胚を移し、フィルボイドバブル形成。 </li><li>メディア溢れを回避または除去する＃0カバーガラスでカバーしています。マニキュアでそれを封印。 </li><li> 633励起フィルターやレーザーラインを装備した蛍光顕微鏡で標本の画像を取得します。 注：発光フィルタは、メチルグリーンの677発光極大をカバーする必要があります。 </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.

厚い試料の核染色。本明細書に説明されたプロトコルは、全胚における深部構造の均一な染色を達成することができます。レンズの開発48 HPFのゼブラフィッシュ胚が均一に標識されている核の形態（ 図1）。 メチルグリーンDNA染色は、有糸分裂像やアポトーシス核の識別（ 図2A）のような、細胞周期依存性の形態学的な違いの識別を可能にしま?...

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.

胚の蛍光標識でDNA特異的染色メチルグリーンの応用

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

Research

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Developmental Biology

14.0K ビュー.  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.