وأيد هذا العمل أعد الصورة جائزة طب الأطفال الأمريكية الجراحية جمعية مؤسسة (AG) والمعاهد الوطنية للصحة K08DK098271 (AG).

وافقت جامعة ويسكونسن رعاية الحيوان واستخدام اللجنة كافة الإجراءات. 1. إعداد حلول <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام 1X الفوسفات مخزنة المالحة (PBS) كما عازلة تشريح وحل الشطف. </li><li style=";text-align:right;direction:rtl"> إعداد 30٪ سكروز عن طريق وزن 30 غرام من السكروز ومكان في زجاجة. إضافة 99 مل من برنامج تلفزيوني 1X وإضافة 1 مل 10٪ أزيد الصوديوم. تخلط جيدا حتى يذوب كل من السكروز. تخزينها في 4 درجات مئوية حتى المطلوبة. </li><li style=";text-align:right;direction:rtl"> إعداد عرقلة الحل عن طريق خلط 1X PBS، 3٪ ألبومين المصل البقري (BSA) و 0.1٪ تريتون-X-100. تخلط جيدا وتخزينها في الثلاجة لحين الحاجة إليها. </li><li style=";text-align:right;direction:rtl"> إعداد 8٪ امتصاص العرق (PFA) حل في برنامج تلفزيوني 1X عن طريق وزن كمية مناسبة من PFA في برنامج تلفزيوني 1X ثم احتضان عند 65 درجة مئوية حتى يذوب تماما. متجر في 25 مل قسامات في -20 ° C الفريزر لحين الحاجة إليها. تمييع إلى 4٪ PFA في برنامج تلفزيوني 1X في يوم من الاستخدام. </li></ol> 2. الأجنة والوتر تشريحأيون <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وفقا لرعاية الحيوان المؤسسية واستخدام اللجنة وافقت البروتوكولات، الموت ببطء توقيت الماوس الحوامل ونقل الرحم إلى 60 مم طبق بتري تحتوي على الجليد الباردة برنامج تلفزيوني 1X. </li><li style=";text-align:right;direction:rtl"> تحت المجهر تشريح باستخدام مقص حاد، وقطع جدار الرحم مفتوح لفضح الأجنة. إزالة الأجنة من الرحم والمكان إلى بيئة نظيفة 60 مم طبق بتري تحتوي على الجليد الباردة برنامج تلفزيوني 1X. </li><li style=";text-align:right;direction:rtl"> الموت ببطء كل ​​الجنين عن طريق قطع الرأس في الجليد الباردة برنامج تلفزيوني 1X. إذا كنت تستخدم الفئران المعدلة وراثيا تحتوي على البروتينات الفلورية، تحت إضاءة مضان، وتحديد الأجنة وراثيا إيجابية. </li><li style=";text-align:right;direction:rtl"> تشريح الجهاز الهضمي من كل جنين باستخدام مجهر تشريح. باستخدام ملقط غرامة، توجيه الجنين مثل أن الجانب الأيسر ومتجهة لأعلى وعلى الجانب الأيمن هو ضد الجزء السفلي من طبق بيتري. إزالة جدار الجزء العلوي من الجسم من الجنين لفضح الأعضاء الداخلية. إدراج ملقط غرامة بين جدار الجسم الظهري والأعضاء الداخلية. كرتابعنا ملقط ضد بعضها البعض في عمل قطع مثل مقص لإزالة الأعضاء الداخلية من الجنين. </li><li style=";text-align:right;direction:rtl"> مزيد من دون تشريح الجهاز الهضمي بعيدا عن الأجهزة المحيطة ومن ثم وضع كل الجهاز الهضمي في أنبوب microcentrifuge 1.5 مل تحتوي على الجليد الباردة برنامج تلفزيوني 1X. </li></ol> 3. تثبيت للGI شيتاغونغ <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> شطف كل الجهاز الهضمي 3 مرات مع الثلج الباردة برنامج تلفزيوني 1X ثم قم باستبدال مع 4٪ PFA. إصلاح مساحات GI في 1.5 مل أنابيب microcentrifuge على منصة هزاز في RT لمدة 1.5 ساعة. شطف مساحات GI 3 مرات لمدة 5 دقائق على RT ثم لمدة 1 ساعة على منصة هزاز. في هذه المرحلة، وتخزين مساحات GI في 4 ° C في 30٪ سكروز في برنامج تلفزيوني 1X تحتوي على 0.1٪ أزيد الصوديوم لحين الحاجة إليها. ملاحظة: بدلا من ذلك، تخزين الأجنة في 30٪ سكروز لمدة تصل إلى واحد من دون أي تأثير على سلامة الأنسجة. تخزين العينات في 30٪ سكروز يسمح معالجة لاحق من العينات إما لالمناعية أو إلى أكتوبر لالبرد sectioniنانوغرام. بدلا من ذلك، المضي قدما في بروتوكول المناعية المفصلة أدناه. </li></ol> 4. بروتوكول المناعية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إذا تم تخزينها العينات في 30٪ سكروز، شطف لهم 3 مرات لمدة 20 دقيقة في برنامج تلفزيوني 1X على منصة هزاز. </li><li style=";text-align:right;direction:rtl"> وضع مساحات GI إلى عرقلة الحل على منصة هزاز ل1H في RT. </li><li style=";text-align:right;direction:rtl"> إزالة حل الحجب واحتضان مساحات GI مع كمية مناسبة من الأجسام المضادة الأولية مخففة في عرقلة الحل إما 4 ساعة على RT أو O / N عند 4 درجات مئوية على منصة هزاز. استخدام 1: 1000 تخفيف البشرية الأضداد المضادة هو جين تاو (المصل تم الحصول عليها من المريض)، 1: 1000 تخفيف من الدجاج مكافحة الأخضر بروتين فلوري (GFP) الأجسام المضادة و 1: 100 التخفيف من الماعز الأجسام المضادة لمكافحة الدردشة. ملاحظة: علينا الاستفادة من الأجسام المضادة لمكافحة البشري هو جين تاو التي تم الحصول عليها محليا من المريض، ومع ذلك، هي أضداد هو جين تاو المتاحة تجاريا، على سبيل المثال، والماوس المضادة للهو جين تاو، (استخدام في 1: 500 تمييع). </li><li style=";text-align:right;direction:rtl"> شطف مساحات GI في برنامج تلفزيوني 1X3 مرات لمدة 5 دقائق ثم لمدة 1 ساعة على RT على منصة هزاز. </li><li style=";text-align:right;direction:rtl"> استبدال برنامج تلفزيوني 1X مع الأجسام المضادة الثانوية المخفف 1: 500 في عرقلة الحل على منصة هزاز إما 4 ساعة على RT أو O / N عند 4 درجات مئوية. استخدام حمار مكافحة الإنسان وأمين صبغ رد الفعل مثل Dylight، حمار Cy2 مكافحة الدجاج وحمار المضادة للماعز Cy5. إذا باستخدام الماوس المضادة للهو الضد ثم استخدم 1: 500 التخفيف من حمار مكافحة الماوس أمين صبغ رد الفعل 405. ملاحظة: كثافة GFP الذاتية وtdTomato التعبير ووضوح المناعية الزيادة مع التقدم في العمر التنموي. نحن immunostain عادة الشجاعة الجنينية بشكل فردي في 0.2 مل الأنابيب مع 150 ميكرولتر من الحل تلطيخ من أجل الحد من حجم الأجسام المضادة المستخدمة وأيضا لضمان كفاءة تلطيخ من الأنسجة. </li><li style=";text-align:right;direction:rtl"> شطف مساحات GI في برنامج تلفزيوني 1X 3 مرات لمدة 5 دقائق ثم لمدة 1 ساعة على RT على منصة هزاز. </li><li style=";text-align:right;direction:rtl"> ضع بضع قطرات من مضان تصاعد المتوسطة مع دابي على شريحة زجاجية. تزج هيئة تنظيم الاتصالات GIط م في المتوسط ​​مضان تركيب وإضافة غطاء زجاجي مباشرة على الجزء العلوي من الأنسجة. مضان تصاعد -G المتوسطة هو مركب للذوبان في الماء الذي يوفر ختم شبه دائم. ملاحظة: استخدام مضان المتوسطة المتزايدة مثل Vectashield وهو الجلسرين المستندة إلى تصاعد المتوسط ​​الذي يمنع يتلاشى وphotobleaching من الأجسام المضادة. كل من هذه المنتجات تمكين الأنسجة ليتم تخزينها لفترات طويلة من الزمن في 4 ° C. </li><li style=";text-align:right;direction:rtl"> التقاط الصور من كل من fluorophore باستخدام مجهر متحد البؤر. استخدام موجات الإثارة لfluorophores والفلاتر المستخدمة الواردة في الجدول 3. </li><li style=";text-align:right;direction:rtl"> لتحليل صورة بمساعدة الكمبيوتر باستخدام البرمجيات المناسبة تبعا لنوع من متحد البؤر استخدامها لالتقاط الصور. </li></ol>

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The authors have nothing to disclose.

وقد أظهرت مختبرنا وغيرها من الجهات التي عيوب المعوية في HSCR لا تقتصر على القولون بانعدام العقد ولكن تمديد قريب، حتى في الأمعاء الدقيقة عقدي 5،15،16. وتشمل هذه التغيرات تغيرات في الخلايا العصبية ENS الكثافة والعصبي النمط الظاهري وربما تمثل dysmotility التي لوحظت في المرض...

ويعمل الجهاز العصبي المعوي (ENS) التي تسيطر على الحركة، وامتصاص المواد الغذائية، وتدفق الدم المحلي، أمر ضروري في الحياة 1. يتم تشكيل ENS من قبل خلايا قمة العصبية (NCC) التي تتكاثر، الهجرة واستعمار القناة الهضمية، حيث تفرق في العقد تحتوي على الخلايا العصبية والخلايا الدبقية. مرض هيرشسبرونغ (HSCR، الانترنت وراثة مندلية في مان)، وهو اضطراب خلقي multigeneic مع حدوث 1 في 4000 ولادة حية، يمكن اعتبار المرض prototypic لدراسة تشكيل ENS تعطلت. في HSCR، NCC تفشل في الهجرة إلى استعمار وأطوال مختلفة بين المعى المؤخر القاصي 2. بالإضافة إلى ذلك، ترتبط الجهاز الهضمي المشتركة الأخرى (GI) وجود عيوب التنموية في عدد من الأطفال، مثل التشوهات الشرجية، atresias المعوية، واضطرابات الحركة مع اضطرابات في وظائف ENS الأساسية، وعلى الأرجح يرتبط خفية، لا تقدر حق قدرها، والتغيرات التشريحية وتغييرات وظيفية في3-6 ENS. لذلك، قد التقنيات التي تسمح لنا لفهم المحددات التنموية لتشكيل ENS تسليط الضوء على المرضية والعلاج المحتملة للاضطرابات الجهاز الهضمي للأطفال. وبعد الهجرة والاستعمار، NCC يفرق في الخلايا العصبية مع علامات محددة لالنمط الظاهري العصبي بهم. تتكون الخلايا العصبية كوليني ما يقرب من 60٪ من الخلايا العصبية المعوية 7، ويمكن الكشف عنها بواسطة تلطيخ للأسيتيل كولين (الدردشة)، الانزيم توليف لالناقل العصبي الأسيتيل كولين مثير. تاريخيا، يحاول تصور ومرتبك الخلايا العصبية الكوليني التي كتبها اختلاف مستضد خصوصية الأجسام المضادة الموجهة ضد النظام العصبي المركزي (CNS) دردشة مقابل الجهاز العصبي المحيطي (السندات الإذنية) دردشة 8-10. ومع ذلك، الأجسام المضادة الموجهة ضد المشيمة دردشة تعترف كل من دردشة المركزية والطرفية 11-13، ولدينا تقنيات وصفها مؤخرا التي تسمح للvisualization من ENS الخلايا العصبية الكوليني مع حساسية عالية في التنمية في وقت سابق من تحقق مع خطوط مراسل دردشة 14. هنا، نقدم تقنية لتشريح، وتحديد والمناعية من الفئران الجنينية GI الجهاز لتصور ENS العصبي التعبير في الخلايا العصبية. لهذه الدراسات، ولقد استخدمت الفئران دردشة لجنة المساواة العرقية تزاوج مع R26R: الحيوانات tdTomato لإنتاج دردشة لجنة المساواة العرقية؛ R26R: floxSTOP الفئران tdTomato (كما هو موضح دردشة لجنة المساواة العرقية tdTomato في جميع أنحاء المخطوط): floxSTOP. ثم تم تزاوج هذه الحيوانات مع متماثل الفئران مراسل دردشة GFP، للحصول على الفئران التعبير عن كل صحفيين الفلورسنت التي تكشف عن التعبير دردشة 14. هذه الحيوانات مراسل هما على خلفية C57BL / 6J ومتاحة تجاريا (مختبرات جاكسون، بار هاربور، ME).

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			<td>Phosphate Buffered Saline</td>
			<td>Oxoid</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Fisher</td>
			<td>BP9221</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>BP1605</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dishes</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence scope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine science tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech, Birmingham, AL</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>16004-330</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>Nikon A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Elements</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

immunostaining to visualize murine enteric nervous system development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

وصفناها سابقا جيل من الفئران التعبير عن كل GFP وtdTomato للصحفيين الفلورسنت التي تكشف عن التعبير دردشة 14. باختصار، كانت تزاوج الفئران دردشة لجنة المساواة العرقية مع R26R: floxSTOP: الحيوانات tdTomato لإنتاج دردشة لجنة المساواة العرقية؛ R26R: الفئران tdTomato</e...

Watch this Scientific Journal Video about Immunostaining to Visualize Murine Enteric Nervous System Development at JoVE.com

المناعية لتصور تطوير الجهاز العصبي المعوي الفئران | علم الأحياء التنموي | فيديو

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

المناعية لتصور الفئران المعوية العصبي تطوير النظام

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

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11.3K Views.  University of Wisconsin School of Medicine and Public Health. The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Video: Immunostaining to Visualize Murine Enteric Nervous System Development

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

الأمثل<em&gt; السابقين البيضة</em&gt; التثقيف من الفرخ الأجنة إلى مراحل متقدمة من التطوير

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

باستخدام ورقة ضوء مضان المجهري لصورة التنمية الزرد العين

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

تقنية زرع الثدي الثديية الرواية جديدة لتصور الجيل سفينة من الخلايا الجذعية البطانية الأوعية الدموية

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

تصوير مرور الوقت فوتون متعددة لتصور التنمية في الوقت الحقيقي: التصور لترحيل الخلايا العصبية كريست في الأجنة الزرد

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

أسلوب الثقافة أورجانوتيبيك لدراسة تطوير الأنسجة الجنينية الدجاج

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

التحليل الحسابي ل Germline ايليجانس كاينورهابديتيس دراسة توزيع الأنوية، والبروتينات، وسيتوسكيليتون

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

زراعة أنسجة المبيض تصور الظواهر في المبيض الماوس

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila larvae, and fish, which all engage in undulatory movements (like crawling or swimming) as their primary mode of locomotion. However, a more sophisticated understanding of the individual motor neuron (MN) specification&#8212;at least in terms of informing disease therapies&#8212;demands an equally tractable system that better models the complex appendage-based locomotion schemes of vertebrates. The adult Drosophila locomotor system in charge of walking meets all of these criteria with ease, since in this model it is possible to study the specification of a small number of easily distinguished leg MNs (approximately 50 MNs per leg) both using a vast array of powerful genetic tools, and in the physiological context of an appendage-based locomotion scheme. Here we describe a protocol to visualize the leg muscle innervation in an adult fly.

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps.

تصور المورفولوجية الساق الحركية محاور عصبية من خلال بشرة الكبار

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

نظام فحص تكوين الأوعية ثلاثي الأبعاد باستخدام Spheroids الثقافة المشتركة التي شكلتها الخلايا المكونة للمستعمرة البطانية والخلايا الجذعية Mesenchymal

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

التهجين الكامل في الموقع في أجنة حمار وحشي وأنبوب تشكيل اساي في iPSC-ECs لدراسة دور Endoglin في التنمية الوعائية

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...