Name: application of the dna-specific stain methyl green in the fluorescent labeling of embryos
Uploaded: 2015-05-02T14:00:00.000+00:00
Duration: 5 min 59 s
Description: Watch this Scientific Journal Video about Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos at JoVE.com

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.

<html> 1. تنقية للصبغ <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد محلول مائي 4٪ من اللون الأخضر الميثيل عن طريق إذابة 0.4 غرام من مسحوق صمة عار في 10 مل من الماء المقطر. تخلط جيدا حتى يذوب تماما. </li><li style=";text-align:right;direction:rtl"> تحت الدخان غطاء محرك السيارة، ومزجها مع لا يقل عن 2 أجزاء من الكلوروفورم. من المهم التحقق مسبقا ما إذا كانت الأنابيب هي مقاومة الكلوروفورم. </li><li style=";text-align:right;direction:rtl"> تخلط جيدا وأجهزة الطرد المركزي لمدة 1 دقيقة في 2000 x ج لتسريع مرحلة الانفصال. </li><li style=";text-align:right;direction:rtl"> بعد الطرد المركزي، ويتم الحصول على مرحلة العليا المائية مع الأخضر الميثيل ومرحلة العضوية أقل مع الكلوروفورم والمعتاد الملوثات الكريستال البنفسجي. </li><li style=";text-align:right;direction:rtl"> استرداد المرحلة العليا وكرر هذه الخطوة حتى تظهر المرحلة الدنيا خالية تماما من الكريستال البنفسجي. </li><li style=";text-align:right;direction:rtl"> في النهاية، يتم استرداد المرحلة العليا والمخفف في الماء لتركيز النهائي من 2٪ الأخضر الميثيل. هذا المحلول مستقر على طاولة العمل لشهور وليس من الضروري أن تكون محمية من الضوء. </li></ol>&quot;jove_title&quot;&gt; 2. الفلورسنت تلطيخ النووية من الجامعة الزرد الأجنة باستخدام الميثيل الخضراء <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إصلاح الأجنة بين عشية وضحاها في 4٪ فورمالدهايد (من امتصاص العرق) في الفوسفات مخزنة المالحة (PBS). ملاحظة: هذا يمكن أن يستغرق من بضع ساعات ليلة وضحاها اعتمادا على سمك الأجنة. في المثال، فإننا الثابتة 48 ساعة بعد الإخصاب (HPF) الأجنة الزرد بين عشية وضحاها. </li><li style=";text-align:right;direction:rtl"> غسل الأجنة ثلاث مرات في برنامج تلفزيوني-T (1٪ تريتون X-100 في برنامج تلفزيوني) لمدة 5 دقائق. تركيز مرتفع من المنظفات permeabilizes بشرة. </li><li style=";text-align:right;direction:rtl"> إعداد 1: 5،000-1: 10،000 حل الأخضر الميثيل (من 2٪ محلول المخزون) في برنامج تلفزيوني-T. وجود المنظفات أثناء الحضانة مفيد لعينات سميكة، مثل الأجنة الزرد واليرقات. </li><li style=";text-align:right;direction:rtl"> احتضان الأجنة في حل للا يقل عن 6 ساعة عند 4 درجات مئوية، مع هزاز لطيف. مرة أخرى، سمك الجنين المعلمة معايرة لمدة الحضانة. </li><li style=";text-align:right;direction:rtl"> غسل الأجنة 3 تيموفاق مع برنامج تلفزيوني لمدة 30 دقيقة لإزالة الزائدة المنظفات. الميثيل مخضر واضح فقط عند منضمة إلى الحمض النووي، وبالتالي الخلفية من جزيء غير منضم لا يكاد يذكر. </li><li style=";text-align:right;direction:rtl"> جبل على الشريحة حفرها أو غرفة محلية الصنع مع الحل تركيب (75٪ الجلسرين، 0.1 M تريس · حمض الهيدروكلوريك درجة الحموضة 8). ملاحظة: إذا كان تلطيخ النووي التي يتعين القيام بها بعد immunolabeling، يمكن إدراجها الأخضر الميثيل في حل المتصاعدة في تركيز العمل، دون الحاجة إلى غسله. </li><li style=";text-align:right;direction:rtl"> مخزن في الثلاجة أو بدء التصوير مباشرة. أداء التصوير مع متحد البؤر المسح بالليزر (أو مضان آخر) المجهر مع الإثارة في 633 نانومتر، والانبعاثات مجموعة المرشحات للتسجيل في 650-750 نانومتر مع الأخذ بعين الاعتبار أن الميثيل تصل الانبعاثات الأخضر الحد الأقصى في 677 نانومتر. ملاحظة: ونحن نقدم البيانات المقاسة من الميثيل الشخصي الانبعاثات الأخضر، ليتم تحميلها في البرنامج الاستحواذ، كمواد إضافية. </li></ol> 3. نيون سانت النوويةaining من الجامعة الفرخ الأجنة باستخدام الميثيل الخضراء <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> احتضان بيض الدجاج المخصب إلى مرحلة الاهتمام. إصلاح الأجنة بين عشية وضحاها في 4٪ فورمالدهايد (من امتصاص العرق) في برنامج تلفزيوني في 4 درجات مئوية. غسل الأجنة 3 مرات في برنامج تلفزيوني-T لمدة 2 ساعة. </li><li style=";text-align:right;direction:rtl"> إعداد 1: 5،000-1: 10،000 حل الأخضر الميثيل (من 2٪ محلول المخزون) في برنامج تلفزيوني-T. احتضان الأجنة في حل تلطيخ بين عشية وضحاها في 4 درجات مئوية. غسل الأجنة 3 مرات في برنامج تلفزيوني، 1 ساعة لكل منهما. جبل في غرفة مع 75٪ الجلسرين، 0.1 M تريس، ودرجة الحموضة 8. </li></ol> 4. التصوير Fluorescently المسمى النواة في الأجنة كامل محمولة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد غرفة التصوير من خلال إصرارها عدة طبقات من شريط كهربائي على شريحة المجهر وبعناية قطع حفرة في ذلك مع مشرط لوضع الأجنة داخل. وهذا يمنع الأجنة من سحق أثناء التصوير. </li><li style=";text-align:right;direction:rtl"> نقل بعناية الأجنة إلى غرفة التصوير وملء الغرفة إلى الأعلى مع 75٪ الجلسرين، 0.1 M تريس، حمض الهيدروكلوريك، ودرجة الحموضة = 8 لتشكيل فقاعة باطلة. </li><li style=";text-align:right;direction:rtl"> مع تغطية # 0 ساترة تجنب أو القضاء تفيض المتوسط. ختم ذلك بطلاء الأظافر. </li><li style=";text-align:right;direction:rtl"> الحصول على صور من العينة على المجهر مضان مجهزة تصفية 633 الإثارة أو خط ليزر. ملاحظة: يجب أن المرشحات الانبعاثات تغطية الحد الأقصى 677 انبعاث الأخضر الميثيل. </li></ol>

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	<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.

تلطيخ النووي من عينات سميكة. بروتوكول الموصوفة هنا يسمح لتحقيق تلطيخ متجانس من البنى العميقة في أجنة كاملة. وصفت عدسة النوع النامية أنويتها 48 HPF الأجنة الزرد متجانس (الشكل 1). الميثيل تلطيخ DNA الأخضر يسمح تمييز من دورة الخلية الاختلافات الشكلية التي تعتمد مثل التعرف على الأشكال الإنقسامية أو نوى أفكارك (الشكل 2A). هياكل النووية الدقيقة بدقة عالية واضحة أيضا كما هو مبين لنوى البشرة من الأجنة الزرد (الشكل 2B). الميثيل الأخضر مقاوم للphotobleaching من تحت كثافة عالية الإشعاع لفترات طويلة من الزمن. عندما المشع مع ضوء الليزر 633 نانومتر في نفس كثافة، الميثيل تلطيخ النووي الأخضر لا التبييض خارج كما هو الحال تقاسم صمة عار DNA محددة الخصائص الطيفية أخرى مماثلة، TO-PRO-34. وقد تم اختيار طائرة مبائر من شبكية ككل محمولة على الجنين الزرد والتكبير مع الهدف 63X 1.4NA لتحقيق إشعاع عالية. عند تصغيرها، وعينات ملطخة TO-PRO-3 أظهرت تبيض في غضون 60 ثانية من الإشعاع المستمر (الشكل 3، والألواح العليا)، في حين أظهرت عينات ملطخة الأخضر الميثيل توجد أدلة واضحة على photobleaching من حتى في 120 ثانية في نفس الظروف (الشكل 3، والألواح أقل). كانت الصور المكتسبة في مجال البرمجيات اللصف مزيد من معالجة (السطوع / التباين؛ وإعادة الإعمار 3D، الحد الأقصى الإسقاط كثافة) باستخدام فيجي البرمجيات مفتوحة المصدر (http://fiji.sc/). <img alt="الشكل 1" src="/files/ftp_upload/52769/52769fig1.jpg" /> الشكل 1: تلطيخ النووي متجانس من البنى العميقة مع الأخضر الميثيل تطوير عدسة ل48 HPF الجنين الزرد ملطخة الأخضر الميثيل (MG) ​​في س ض الإسقاط<html> و 20 طائرة مبائر. تم counterstained الأجنة لF-الأكتين مع phalloidin TRITC مترافق (فال). شريط النطاق: 15 ميكرون. <img alt="الرقم 2" src="/files/ftp_upload/52769/52769fig2.jpg" /> الشكل 2: التشكل النووي وقرار النووية الدقيقة مع الميثيل الأخضر تلطيخ A) الأشكال التضاريسية النووية مختلفة في البشرة من 48 HPF الجنين الزرد. MG والأخضر الميثيل. تم counterstained العينات للF-الأكتين مع TRITC-phalloidin. رأس السهم: الخلية الإنقسامية. إسقاط الحد الأقصى للكثافة 10 طائرات مبائر. B) الفرخ الجنين لوحة العصبية وصفت في نفس الطريق، وتظهر الخلايا الإنقسامية (السهام) ونوى تغلظية (السهام). طائرة واحدة مبائر. C) ثلاث طائرات متحد البؤر واحدة من الزرد نواة الجنين البشرة في تضخم عالية، مما يدل على درجة عالية من الدقة النووية الدقيقة. أشرطة النطاق: A، 15 ميكرون. B، 5 ميكرون. C، 1 ميكرون. -page = &quot;دائما&quot;&gt; <img alt="الشكل (3)" src="/files/ftp_upload/52769/52769fig3.jpg" /> تم تبييض صمود الخضراء الميثيل في إطار الإثارة مستمرة TO-PRO-3 الملون ونوى 48 HPF شبكية العين الزرد على 60 ثانية من الإثارة مستمرة في 633 نانومتر (لوحات العليا): الشكل (3). في ظل نفس الظروف، لم جنين الملون الأخضر الميثيل (MG) لا تظهر تبيض ملموس، حتى عند تكرار مدة التعرض (لوحات أقل). وقدم تبيض والاستحواذ عن طريق المسح الضوئي في طائرات واحدة، في 8000 هرتز، و 30٪ قوة الليزر وخط (X2) والإطار (X4) المتوسط. شريط النطاق: 30 ميكرون. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td> الطول الموجي (نانومتر) </td><td> الانبعاثات في المئة </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><tdمحاذاة = &quot;الحق&quot;&gt; 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 = &quot;الحق&quot;&gt; 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= &quot;الحق&quot;&gt; 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= &quot;الحق&quot;&gt; 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> المواد التكميلية 1: انبعاث الطيف من الميثيل تلطيخ النووي الأخضر تحت 633 نانومتر الإثارة.

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.

تطبيق للDNA محددة وصمة عار الميثيل الأخضر في بطاقات المواد الفلورية الأجنة

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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Developmental Biology

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.

فهم توالد المبكر باستخدام المبسطة<em&gt; في الموقع</em&gt; بروتوكول التهجين في<em&gt; القيطم</em

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

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.

تحليل كمي للتعبير البروتين لدراسة النسب مواصفات في ماوس سابق للانغراس الأجنة

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.

متعددة الهدف اللونية الجامع جبل<em&gt; في الموقع</em&gt; التهجين لمقارنة الجينات مجالات التعبير في<em&gt; ذبابة الفاكهة</em&gt; الأجنة

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.

مضان المجهر المعيشة أو الثابتة والملون على أساس ورقة الخفيفة<em&gt; خنفساء الدقيق الحمراء</em&gt; الأجنة

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

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

وضع العلامات واحدة من الخلايا في الجهاز العصبي المركزي<em&gt; ذبابة الفاكهة الدروسوفيلا</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Neuroscience

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

ثنائي الفوتون المستندة إلى تنشيط ضوئي في الأجنة الحية اسماك الزرد

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

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.

تصور الاتجار المعتمد على الهيكل الخلوي للعضيات المحتوية على الدهون في أجنة ذبابة الفاكهة

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.

جبل به الجامعة في الموقع تهجين ارتباط البيولوجيا الجزيئية والعضوي

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

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

جبل كله<em&gt; في الموقع</em&gt; التهجين من E8.5 لأجنة الفئران E11.5

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

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.

وسم الخلية وحقن في تطوير الجنينية الماوس قلوب

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

تطبيق للDNA محددة وصمة عار الميثيل الأخضر في بطاقات المواد الفلورية الأجنة

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فهم توالد المبكر باستخدام المبسطة

تحليل كمي للتعبير البروتين لدراسة النسب مواصفات في ماوس سابق للانغراس الأجنة

متعددة الهدف اللونية الجامع جبل

مضان المجهر المعيشة أو الثابتة والملون على أساس ورقة الخفيفة

وضع العلامات واحدة من الخلايا في الجهاز العصبي المركزي

ثنائي الفوتون المستندة إلى تنشيط ضوئي في الأجنة الحية اسماك الزرد

تصور الاتجار المعتمد على الهيكل الخلوي للعضيات المحتوية على الدهون في أجنة ذبابة الفاكهة

جبل به الجامعة في الموقع تهجين ارتباط البيولوجيا الجزيئية والعضوي

جبل كله

وسم الخلية وحقن في تطوير الجنينية الماوس قلوب