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. Purification of the Dye
<ol>
	<li>Prepare a 4% aqueous solution of methyl green by dissolving 0.4 g of stain powder in 10 ml distilled water. Mix thoroughly until completely dissolving it.</li>
	<li>Under a fume-hood, mix it with at least 2 parts of chloroform. It is important to check beforehand whether the tubes are chloroform resistant.</li>
	<li>Mix thoroughly and centrifuge for 1 min&#160;at 2,000 x g to accelerate phase separation.</li>
	<li>After centrifugation, an upper aqueous phase with methyl green and a lower organic phase with chloroform and the usual contaminant crystal violet are obtained.</li>
	<li>Recover the upper phase and repeat this step until lower phase appears completely devoid of crystal violet.</li>
	<li>At the end, the upper phase is recovered and diluted in water to a final concentration of 2% methyl green. This stock solution is stable on the workbench for months and does not need to be protected from light.</li>
</ol>
2. Fluorescent Nuclear Staining of Whole Zebrafish Embryos using Methyl Green
<ol>
	<li>Fix the embryos overnight in 4% formaldehyde (from paraformaldehyde) in phosphate buffered saline (PBS). 
	NOTE: This can take from a few hours to overnight depending on the thickness of the embryos. In the example, we fixed 48 hr&#160;post-fertilization (hpf) zebrafish embryos overnight.</li>
	<li>Wash the embryos three times in PBS-T (1% Triton X-100 in PBS) for 5 min. The elevated concentration of detergent permeabilizes the cuticle.</li>
	<li>Prepare a 1:5,000-1:10,000 solution of methyl green (from 2% stock solution) in PBS-T. The presence of detergent during the incubation is useful for thick specimens, such as zebrafish embryos and larvae.</li>
	<li>Incubate the embryos in the solution for at least 6 hr&#160;at 4 &#176;C, with gentle rocking. Again, the thickness of the embryo is the calibrating parameter for the length of the incubation.</li>
	<li>Wash the embryos 3 times with PBS for 30 min to remove the detergent excess. Methyl green fluorescence is only evident when bound to DNA, hence background from the unbound molecule is negligible.</li>
	<li>Mount on an excavated slide or a homemade chamber with mounting solution (75% glycerol, 0.1 M Tris&#183;HCl pH 8). 
	NOTE: If the nuclear staining is to be performed after immunolabeling, methyl green can be incorporated to the mounting solution at the working concentration, with no need to wash it away.</li>
	<li>Store refrigerated or start imaging immediately. Perform imaging with a laser scanning confocal (or other fluorescence) microscope with excitation at 633 nm and emission filters set to register at 650-750 nm taking into account that methyl green emission reaches its maximum at 677 nm. 
	NOTE: We provide the measured data of the methyl green emission profile, to be loaded into the acquisition software, as supplementary material.</li>
</ol>
3. Fluorescent Nuclear Staining of Whole Chick Embryos using Methyl Green
<ol>
	<li>Incubate fertilized hen eggs to the stage of interest. Fix the embryos overnight in 4% formaldehyde (from paraformaldehyde) in PBS at 4 &#176;C. Wash embryos 3 times in PBS-T for 2 hr.</li>
	<li>Prepare a 1:5,000-1:10,000 solution of methyl green (from 2% stock solution) in PBS-T. Incubate the embryos in the staining solution overnight at 4 &#176;C. Wash the embryos 3 times in PBS, 1 hr&#160;each. Mount in chamber with 75% glycerol, 0.1 M Tris, pH 8.</li>
</ol>
4. Imaging Fluorescently Labeled Nuclei in Whole-mounted Embryos
<ol>
	<li>Prepare an imaging chamber by sticking several layers of electrical tape over a microscope slide and carefully cut a hole into it with a scalpel to place the embryos within. This will prevent embryos from crushing during imaging.</li>
	<li>Carefully transfer the embryos to the imaging chamber and fill the chamber to the top with 75% glycerol, 0.1 M Tris-HCl, pH=8 to avoid bubble formation.</li>
	<li>Cover with a #0 coverslip avoiding or eliminating medium overflowing. Seal it with nail polish.</li>
	<li>Acquire images of the specimen on a fluorescence microscope equipped with a 633 excitation filter or laser line. 
	NOTE: Emission filters should cover the 677 emission maximum of methyl green.</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 Hoechst, are excited with UV light and emit in the blue area of the visible spectrum2,3, making them inadequate for imaging of deep structures in whole tissues. Red light penetrates much better into biological samples, making red-emitting fluorophores more suitable for these studies12. Autofluorescence is a common problem in embryonic tissues that can also be overcome by using red-emitting fluorophores5.
We have shown that the very common histological stain methyl green is a highly efficient red-emitting fluorophore when bound to DNA, and it can be applied to the detection of DNA in microscopy, flow cytometry, fluorometry or gel electrophoresis6. The protocol for fluorescent nuclear labeling of whole embryos with methyl green described here is relatively fast and straightforward. It is an inexpensive technique and a stock solution suitable for hundreds of experiments can be prepared with minimal effort in the lab.
The critical steps of this method are stain purification, specimen permeabilization and incubation times for whole embryos. Crystal violet contamination can easily affect methyl green fluorescent properties thus making it essential to have a highly pure methyl green solution. Methyl green is safer than ethidium bromide as it is a non-intercalant dye which cannot enter cells unless when membrane integrity is compromised, and this is the basis for its application as a vital dye. However, when nuclear DNA is to be stained in fixed embryos, it is required that cuticles and cells go through a permeabilization step such as the ones described here, i.e. incubation with a detergent. Thicker embryos require longer incubation times, as methyl green will enter the embryos by diffusion and minimal incubation times described herein can be used as a reference for other types of specimens. Although we have not tested methyl green in thicker diaphanized tissues as whole mounted organs, due to its spectral properties we do not forsee major limitations to its use provided that chemical clarification (i.e. CLARITY, methysalicilate, Sca/e, etc) does not react with methyl green. An interesting point would be to test its suitability for multi-photon excitation, as this illumination allows for deeper specimen penetration. A simple and useful modification of the protocol can be used for cultured cells by incubating them for 5 minutes with methyl green.
Finally, methyl green emission spectrum is relatively narrow when compared with other red-emitting DNA stains (such as propidium iodide), it presents a relatively large Stokes shift (44 nm) and it is extremely resistant to photobleaching. All these characteristics make it ideal for high resolution laser confocal imaging of whole embryos, as it can be combined with several other fluorophores at the same time and it allows for long exposures.

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><tbody><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Methyl green</td><td>Dr. G. Gr&amp;uuml;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&amp;ndash;Tetramethylrhodamine B isothiocyanate</td><td>Sigma-Aldrich</td><td>&lt;br/&gt; P1951&amp;nbsp;</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&amp;nbsp;&amp;nbsp;</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.

Nuclear staining of thick specimens. The protocol herein described allows for the achievement of homogeneous staining of deep structures in whole embryos. Developing lens nuclei form 48 hpf zebrafish embryos are homogeneously labeled (Figure 1).
Methyl green DNA staining allows discrimination of cell cycle dependent morphological differences such as the identification of mitotic figures or apoptotic nuclei (Figure 2A). Subnuclear structures at high resolution are also evident as shown for epidermal nuclei of zebrafish embryos (Figure 2B).
Methyl green is resistant to photobleaching under high intensity irradiation for prolonged periods of time. When irradiated with 633 nm laser light at the same intensity, methyl green nuclear staining does not bleach out as it is the case of another DNA-specific stain sharing similar spectral characteristics, TO-PRO-34. A confocal plane of the retina of a whole-mounted zebrafish embryo was selected and zoomed in with a 63x 1.4NA objective to achieve high irradiation. When zoomed out, the specimens stained with TO-PRO-3 showed bleaching within 60 seconds of continuous irradiation (Figure 3, upper panels), while the specimens stained with methyl green showed no visible evidence of photobleaching even at 120 seconds in the same conditions (Figure 3, lower panels). Images acquired in LASAF software were further processed (brightness/contrast; 3D reconstruction; maximum intensity projection) using Fiji open source software (http://fiji.sc/).
<img alt="Figure 1" src="/files/ftp_upload/52769/52769fig1.jpg" /> 
Figure 1: Homogeneous nuclear staining of deep structures with methyl green. Developing lens of a 48 hpf zebrafish embryo stained with methyl green (MG) in a z-projection of 20 confocal planes. Embryos were counterstained for F-actin with TRITC-conjugated phalloidin (Phal). Scale bar: 15 &#181;m.
<img alt="Figure 2" src="/files/ftp_upload/52769/52769fig2.jpg" /> 
Figure 2: Nuclear morphology and subnuclear resolution with methyl green staining. A) Different nuclear morphologies in the epidermis of a 48 hpf zebrafish embryo. MG, methyl green. Specimens were counterstained for F-actin with TRITC-phalloidin. Arrowhead: mitotic cell. Maximum-intensity projection of 10 confocal planes. B) Chick embryo neural plate labeled in the same way, displaying mitotic cells (arrowheads) and pyknotic nuclei (arrows). Single confocal plane. C) Three single confocal planes of a zebrafish embryo epidermal nucleus at high magnification, showing a high degree of subnuclear resolution. Scale bars: A, 15 &#181;m; B, 5 &#181;m; C, 1 &#181;m.
<img alt="Figure 3" src="/files/ftp_upload/52769/52769fig3.jpg" /> 
Figure 3: Photostability of methyl green under continuous excitation. TO-PRO-3-stained nuclei of a 48 hpf zebrafish retina was bleached upon 60 sec&#160;of continuous excitation at 633 nm (upper panels). Under the same conditions, a methyl green-stained embryo (MG) did not show perceptible bleaching, even when duplicating the exposure time (lower panels). Bleaching and acquisition was made by scanning in single planes, at 8,000 Hz, 30% laser power and line (x2) and frame (x4) averaging. Scale bar: 30 &#181;m.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Wavelength (nm)</td>
			<td>Percent emission</td>
		</tr>
		<tr>
			<td align="right">640</td>
			<td align="right">21.37</td>
		</tr>
		<tr>
			<td align="right">643</td>
			<td align="right">27.5</td>
		</tr>
		<tr>
			<td align="right">646</td>
			<td align="right">36.69</td>
		</tr>
		<tr>
			<td align="right">649</td>
			<td align="right">44.47</td>
		</tr>
		<tr>
			<td align="right">652</td>
			<td align="right">53.92</td>
		</tr>
		<tr>
			<td align="right">655</td>
			<td align="right">61.82</td>
		</tr>
		<tr>
			<td align="right">658</td>
			<td align="right">67.86</td>
		</tr>
		<tr>
			<td align="right">661</td>
			<td align="right">83.2</td>
		</tr>
		<tr>
			<td align="right">664</td>
			<td align="right">85.22</td>
		</tr>
		<tr>
			<td align="right">667</td>
			<td align="right">89.45</td>
		</tr>
		<tr>
			<td align="right">670</td>
			<td align="right">87.33</td>
		</tr>
		<tr>
			<td align="right">673</td>
			<td align="right">92.3</td>
		</tr>
		<tr>
			<td align="right">676</td>
			<td align="right">100</td>
		</tr>
		<tr>
			<td align="right">679</td>
			<td align="right">97.41</td>
		</tr>
		<tr>
			<td align="right">682</td>
			<td align="right">90.34</td>
		</tr>
		<tr>
			<td align="right">685</td>
			<td align="right">83.32</td>
		</tr>
		<tr>
			<td align="right">688</td>
			<td align="right">78.37</td>
		</tr>
		<tr>
			<td align="right">691</td>
			<td align="right">76.09</td>
		</tr>
		<tr>
			<td align="right">694</td>
			<td align="right">73.95</td>
		</tr>
		<tr>
			<td align="right">697</td>
			<td align="right">67.36</td>
		</tr>
		<tr>
			<td align="right">700</td>
			<td align="right">64.4</td>
		</tr>
		<tr>
			<td align="right">703</td>
			<td align="right">61.26</td>
		</tr>
		<tr>
			<td align="right">706</td>
			<td align="right">56.39</td>
		</tr>
		<tr>
			<td align="right">709</td>
			<td align="right">55.06</td>
		</tr>
		<tr>
			<td align="right">712</td>
			<td align="right">49.56</td>
		</tr>
		<tr>
			<td align="right">715</td>
			<td align="right">46.12</td>
		</tr>
		<tr>
			<td align="right">718</td>
			<td align="right">41.4</td>
		</tr>
		<tr>
			<td align="right">721</td>
			<td align="right">38.8</td>
		</tr>
		<tr>
			<td align="right">724</td>
			<td align="right">35.97</td>
		</tr>
		<tr>
			<td align="right">727</td>
			<td align="right">33.27</td>
		</tr>
		<tr>
			<td align="right">730</td>
			<td align="right">30.95</td>
		</tr>
		<tr>
			<td align="right">733</td>
			<td align="right">28.78</td>
		</tr>
		<tr>
			<td align="right">736</td>
			<td align="right">26.28</td>
		</tr>
		<tr>
			<td align="right">739</td>
			<td align="right">24.96</td>
		</tr>
		<tr>
			<td align="right">742</td>
			<td align="right">23.3</td>
		</tr>
		<tr>
			<td align="right">745</td>
			<td align="right">21.12</td>
		</tr>
		<tr>
			<td align="right">748</td>
			<td align="right">19.86</td>
		</tr>
		<tr>
			<td align="right">751</td>
			<td align="right">17.47</td>
		</tr>
		<tr>
			<td align="right">754</td>
			<td align="right">16.14</td>
		</tr>
		<tr>
			<td align="right">757</td>
			<td align="right">14.21</td>
		</tr>
		<tr>
			<td align="right">760</td>
			<td align="right">12.45</td>
		</tr>
		<tr>
			<td align="right">763</td>
			<td align="right">11.49</td>
		</tr>
		<tr>
			<td align="right">766</td>
			<td align="right">9.97</td>
		</tr>
		<tr>
			<td align="right">769</td>
			<td align="right">7.55</td>
		</tr>
		<tr>
			<td align="right">772</td>
			<td align="right">7.75</td>
		</tr>
		<tr>
			<td align="right">775</td>
			<td align="right">6.46</td>
		</tr>
		<tr>
			<td align="right">778</td>
			<td align="right">5.76</td>
		</tr>
		<tr>
			<td align="right">781</td>
			<td align="right">4.38</td>
		</tr>
		<tr>
			<td align="right">784</td>
			<td align="right">2.6</td>
		</tr>
		<tr>
			<td align="right">787</td>
			<td align="right">2.75</td>
		</tr>
		<tr>
			<td align="right">790</td>
			<td align="right">2.27</td>
		</tr>
		<tr>
			<td align="right">793</td>
			<td align="right">1.52</td>
		</tr>
		<tr>
			<td align="right">796</td>
			<td align="right">0</td>
		</tr>
	</tbody>
</table>
Supplementary material 1: Emission spectrum of methyl green nuclear staining under 633 nm excitation.

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.

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.

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

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Biology

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

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

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