접착 챔버스 준비 (전날 사출 / 절개) 접착 분석 챔버스 당신은 종래의 복합 현미경 (무대 위 광학 찾아)이있다면, 그것은 낮은 높이 접착력 챔버는 다음과 같이 할 ncecessary입니다. 거꾸로 - 광학 현미경을 사용하는 경우, &quot;NUNC 연구소 - 테크 Chambered의 Coverglass은&quot;단지 사용하고, 단계 # 1 생략 수 있습니다 - 2. <ol><li> 40 X 24mm 커버 슬립에 &quot;언론 - 투 - 밀봉 실리콘 절연체&quot;중 하나를 첨부합니다. 단단히 깨끗한 benchtop에서 커버 슬립에 절연체를 눌러 절연체가 완전히 다른 공기 방울없이 연결되었는지 확인하기 위해 반대편에서보세요. </li><li> 선택 사항 : 소독하기 위해 드라이주기 왓먼 필터 종이 압력솥과 유리 페트리 접시에 부착 실 놔. RT에게 시원한 시간을 보내세요. </li></ol> 코팅 상공 회의소 <ol><li> sigmacoated 노란색 도움말을 사용하여 autoclaved 부착 챔버의 중심에 cadherin (10mg/ml) 또는 fibronectin (20mg/ml)의 솔루션을 작업의 200ml 비말 놔. 4 시간 RT에서 알을 품다. </li><li> cadherin이나 fibronectin 솔루션을 꺼내서하고 1.5ml 튜브에 전송할 수 있습니다. 이 솔루션은 다음 두 주 동안 활용할 수 있습니다. </li><li> 챔버를 차단하기 위해 MBS - H 1X의 4 % BSA의 500ml 분배. 1 시간 RT에서 알을 품다. </li><li> BSA 솔루션을 대기음. 두번 1X MBS - H의 500ml와 챔버를 씻으십시오. 두 번째 세척 후, (아니 완전히) MBS - H의 대부분을 기음, 4 º C.에서 밀폐된 용기에 보관 삼일 -이 유착 챔버은 다음 2 좋습니다. </li></ol> 해부와 동물 캡 Explants의 해리 <ol><li> 비 - autoclaved 매체 언젠가 dissociated 동물 캡 세포를 방해있는 작은 세균을 지니고 있듯이, autoclaved 1X MBS - H에서 희석 0.1x MBS - H에 주입된 배아를 품어. </li><li> 귀하의 샘플의 번호로 적어도 많은, 60mm 요리 1X 바쓰의 아가로 오스 접시와 CMF - MBS 아가로 오스의 번호판을 확인합니다. 1X 바쓰의 접시에 autoclaved 1X MBS - H 하거라. CMF - MBS 접시 들어, CMF - MBS의 10ml를 부어. </li><li> 배아가 무대 8.5이 될 때, 1X 바쓰의 접시에서 동물 모자를 해부 시작합니다. 10 - 샘플 당 15 explants은 실험을 위해 일반적으로 충분합니다. </li><li> CMF - MBS 판에 동물 캡 explants을 전송합니다. explants가 완전히 dissociated까지 60분 - 45 explants을 품어. 매 15 분 후에 부드러운 agitations은 분리 도움이 될 것입니다. </li></ol> 세포 접착 분석 <ol><li> 세포를 심는 것은 : 위의 섹션, 접착 분석 챔버 및 코팅 회의소의 단계에서 준비한 부착 실로 1X MBS - H의 500ml 분배. CMF - MBS 판에서 sigmacoated와 다진 - 오프 노란색 팁을 P200을 사용하여 부착 챔버에 dissociated 동물 캡 세포를 전송합니다. </li><li> 선택 사항 : 가능하면 신중하게 공기에 세포를 노출하지 않고 약 삼분의이 중간에하는 매체를 빨아. 거의 같은 매체가 충분한 칼슘이 포함되어 있는지 확인하기 위해 신선한 금액 MBS - H, +와 MG 2 +. 추가 </li><li> 90분 - 45 RT에서 알을 품다. 그들은 바닥의 표면에 부착하고 있는지 때때로 세포를 확인합니다. </li><li> 지금 당신은 현미경으로 세포의 사진을 찍을 수 있습니다. </li><li> 그리드 슬라이드 유리에 부착 챔버 놔. 그리드 슬라이드의 모서리에 부착 챔버의 모서리 중 하나를 맞춥니다. 백작과 여러 격자에 부착 세포의 수를 기록합니다. </li><li> 큰 플라스틱 양동이 (예 : 유리 섬유 복합 등)에서 MBS - H의 1L를 붓고. 부드럽게 부착 슬라이드 챔버는 양동이의 하단에 넣어. 저속 (- 60rpm 50)에서 5 분 동안 궤도 통에 회전합니다. </li><li> 이 섹션, 세포 접착 분석의 단계 # 5 한 것으로, 그리드 슬라이드의 같은 코너에 챔버의 동일한 모서리를 정렬, 그리드 슬라이드에 다시 부착 챔버 놔. 당신은 세탁하기 전에 계산이 같은 그리드에 부착 남아 세포의 수를 계산합니다. 세척 후 첨부 남아 세포의 비율을 계산합니다. </li></ol>

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1x MBS-H</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Modified Barth&#x2019;s medium: 1L recipe
5.14g		NaCl
75mg		KCl
0.2g		NaHCO3
0.2g		MgSO4&#x2022;7H2O
60mg		CaCl2&#x2022;2H2O
80mg		Ca(NO3)2&#x2022;4H2O
2.38g		HEPES, free-acid
Adjust the pH to 7.4 with NaOH. Autoclave.
</td>
</tr>
<tr>
<td>CMF-MBS	</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>This is Ca2+/Mg2+-free version of MBS-H. 500ml recipe :
2.57g		NaCl
37.5mg		KCl
0.1g		NaHCO3
1.19g		HEPES, free-acid
Adjust the pH to 7.4 with NaOH. Autoclave.
</td>
</tr>
<tr>
<td>1% agarose/1x Barth&#x2019;s plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>as many as the number of samples</td>
</tr>
<tr>
<td>1% agarose/CMF-MBS plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>as many as the number of samples</td>
</tr>
<tr>
<td>E-Cadherin/Fc chimera </td>
<td>Reagent</td>
<td>Sigma</td>
<td>E-2278</td>
<td>Dissolve 0.1mg of solid E-cadherin in 100ul of 1x MBS-H containing 40% glycerol to make 100x master stock (1.0mg/ml). Store it in &#x2013;20C. Make 1/100 dilution with 1x MBS-H to make 10ug/ml working solution just before you use. You need 200ul per samples of this working solution.</td>
</tr>
<tr>
<td>Fibronectin, 0.1% solution </td>
<td>Reagent</td>
<td>Sigma</td>
<td>F-1141</td>
<td>This pre-made solution can be used as 50x stock. Make 1/50 dilution with 1x MBS-H make 20ug/ml working solution just before you use. You need 200ul per samples of this working solution.
I do NOT recommend using solid Fibronectin, because it is difficult to dissolve at 0.1% concentration to make the stock solution. Although it is more expensive, this pre-made 0.1% solution is better to obtain consistent results.
</td>
</tr>
<tr>
<td>4% BSA /MBS-H</td>
<td>Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>BSA: molecular biology grade fraction V</td>
</tr>
<tr>
<td>Sigmacoted yellow tips </td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>No.1 cover slips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>40x24mm or 60x24mm</td>
</tr>
<tr>
<td>Press-to-seal silicon insulator</td>
<td>Tool</td>
<td>Glace Bio-Labs</td>
<td>JTR20-2.5</td>
<td>2.5mm depth x 20mm diameter </td>
</tr>
<tr>
<td>NUNC Lab-Tek Chambered Coverglass</td>
<td>Tool</td>
<td>Fisher</td>
<td>12-565-471</td>
<td>2wells/slides </td>
</tr>
<tr>
<td>Grid slide glass</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>or Finder graticule for cell counting</td>
</tr>		</tbody>
		</table>
		</div>

dissection of organizer and animal pole explants from xenopus laevis embryos and assembly of a cell adhesion assay

Watch this Scientific Journal Video about Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay at JoVE.com

Cell Cycle and Division | Biology Video Collection

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

자기 눈썹 만든 전문 해부 도구 -이 비디오는 눈썹 나이프의 사용을 포함하여 Xenopus laevis의 배아에서 주최 및 동물 극 explants의 준비에 사용되는 기술을 설명합니다. 유착 분석을 조립을위한 프로토콜은 또한 주요 유착 분자의 존재에 대한 프로브가 표면 주최자 또는 적절한 개발을위한 중요 동물 극 세포에 존재하는, 주어진 것입니다.

주최자 및 동물 폴 Explants Xenopus laevis의 태아로부터 세포 접착 분석의 총회의 해부

dissection-organizer-animal-pole-explants-from-xenopus-laevis-embryos

Research

JoVE Journal

Biology

10.6K 조회수.  University of California, Irvine (UCI). 자기 눈썹 만든 전문 해부 도구 -이 비디오는 눈썹 나이프의 사용을 포함하여 Xenopus laevis의 배아에서 주최 및 동물 극 explants의 준비에 사용되는 기술을 설명합니다. 유착 분석을 조립을위한 프로토콜은 또한 주요 유착 분자의 존재에 대한 프로브가 표면 주최자 또는 적절한 개발을위한 중요 동물 극 세포에 존재하는, 주어진 것입니다.

비디오: Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

6.5 DPC 마우스 태아의 해부

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

연구

생물학

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

플라스틱 삽입 및 Xenopus laevis의 태아의 Sectioning

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

Xenopus laevis 여성의 난자를 구하기

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Xenopus laevis 에그의 작성과 분류는 압축을 풉니다

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Xenopus Laevis의 Oocytes의 Microinjection

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

에있는 동물 캡 이식 (ACT) 어세를 사용하여 조직 결정 Xenopus laevis</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

라이브 셀 이미징 및 배아 상피 세포의 정량 분석 Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

의 얼굴 이식<em&gt;는 Xenopus laevis의</em&gt; 태아

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

의 해부<em&gt;는 Xenopus laevis의</em&gt; 신경 크레스트<em&gt; 시험관</em&gt; 이식편 문화 또는<em&gt; 생체</em&gt; 이식

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus의 번역 손상을 식별 할 수있는 모델로 laevis의

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...