固定<ol><li>在一个小玻璃闪烁瓶转移整个胚胎或解剖植（费舍尔猫＃03 - 339 - 25B）到MEMFA或3.7％FA + PBS。在室温孵育nutator小瓶， 只有 2个小时，而不是不止于此。不要离开2个多小时的胚胎在FA。在FA的潜伏期较长，会使胚胎组织的困难，并导致在检测过程中的抗体差渗透。 </li><li>吸大部分的FA和添加登特的固定液的小瓶（〜4.5毫升）顶部。轻轻颠倒几次小瓶洗登特的胚胎，用另一种新鲜的凹痕4.5毫升交流，并在-20 ° C 孵育至少48小时。隔夜不足够的。这一步使胚胎抗体的渗透性。胚胎可以保持在这几个星期的情况下。 </li></ol>免疫<ol><li>补充水分的标本。在甲醇梯度培养，如下： <table style="width: 171px; height: 119px;" border="0" cellpadding="3"><tbody><tr><td> 75％甲醇/ H 2 O </td><td> 10分钟</td></tr><tr><td> 50％甲醇/ PBS </td><td> 10分钟</td></tr><tr><td> 25％甲醇/ PBS </td><td> 10分钟</td></tr><tr><td> PBS </td><td> 10分钟</td></tr><tr><td> PBS </td><td> 10分钟</td></tr></tbody></table> </li><li> 60毫米塑料菜1.5％琼脂糖/ PBS板。倒入琼脂糖上PBS后，它已成为固体。 </li><li> 半胚胎切片 ：将在1.5％琼脂糖/ PBS板的胚胎。一双镊子抓住一个胚胎，并削减了一半的胚胎，用薄薄的刀片中心。选择，您已成功地削减了一半，直切平面的。 <img src="/files/ftp_upload/old/188_2008_7_10_3/188_figure-01.jpg" alt="图1" width="591" height="193" /> </li><li>平分胚胎转移到与PBT的玻璃闪烁瓶。交流4.5毫升的20％山羊血清+ PBT。在室温下孵育2小时上nutator（阻塞）。 </li><li>准备适当稀释，在PBT的主要抗体。这每样本0.5毫升是必要的。例如，我用1 / 200稀释的兔抗c - cadherin的多克隆研究在5mm的塑料型材的C - cadherin的亚细胞定位。 </li><li>吸出阻塞的解决方案，并添加稀释的抗体0.5毫升。在泡沫塑料管持有人站立位置的小瓶。在nutator 4 º C孵育过夜。 </li><li>将稀释的抗体（这种稀释的抗体可以保存并在未来1-2周重复使用）。与PBT的4.5毫升洗4次（每次40-60分钟）在室温下。 </li><li>交换的1 / 100稀释生物素标记的第二抗体在PBT0.5毫升。在nutator 4 º C孵育过夜。用铝箔盖住，以防止光素的小瓶。 </li><li>从这一点来说， 应采取保护光，即样品，保留下一些盖或铝箔的小瓶。 </li><li>删除中学生物素的抗体（这也可以在未来的1-2周重复使用）。洗或PBT4.5毫升4倍（40-60分钟）在室温。 </li><li>交换的1 / 200稀释的荧光标记（FITC，德克萨斯红等），在PBT链霉0.5毫升。在nutator 4 º C孵育过夜。 </li><li>卸下的链霉（可重复使用，这太）。与PBT的4.5毫升洗3次（15 - 30分钟）在室温下。 </li><li> 30分钟，在室温下固定在3.7％FA + PBS4.5毫升胚胎。 </li><li>与PBS4.5毫升洗两次。 </li><li>脱水标本。孵育在乙醇分级系列，如下： <table style="width: 172px; height: 80px;" border="0"><tbody><tr><td> 30％的乙醇/ H 2 O </td><td> 10分钟</td></tr><tr><td> 50％乙醇/ H 2 O </td><td> 10分钟</td></tr><tr><td> 75％乙醇/ H 2 O </td><td> 10分钟</td></tr><tr><td> 100％乙醇</td><td> 10分钟</td></tr></tbody></table> </li><li>试样可存储100％乙醇在4 º C具有保护盖由轻。 </li></ol>浸润<ol><li> Technovit 7100渗透混合。我通常在50ml的锥形管的基液15毫升，添加固化剂150毫克的我​​，并为15分钟nutator摇摆混合。这种渗透组合，可以保持在4 ° C至一个月。 </li><li>渗透与渗透混合标本，通过交流与Technovit渗透的混合/乙醇组合分级系列，如下： <ol><li> Technovit /乙醇 - 1小时50％ </li><li> 100％Technovit - 1小时 </li><li> 100％Technovit - 隔夜 </li></ol></li><li>该specimen可以在100％的渗透混合存储长达一个月在4 ° C。样品放在封面，以保护他们的光。 </li></ol>嵌入<ol><li> Technovit 7100嵌入组合：以0.75毫升渗透在1.5毫升管组合，并添加固化剂二50毫升。颠倒几次管混合。 </li><li>用塑料移液管，取一个试样渗透到0.5ml薄壁PCR管中过夜。删除的大部分上清。 </li><li>添加在本节步骤＃1，“嵌入”，在试样上的嵌入组合0.25毫升。东方平分胚胎面临的试管底部夹层平面，轻轻地用钝尖清扫针（木处理清扫针）轻推。 </li><li>关闭盖子，并在黑暗的地方约四小时，孵化管允许的塑料聚合。 </li><li>请Technovit 3040组合。称量皿中以1克的粉末，而添加的液体0.5毫升。随即，混合粉末和液体使用bluetip。倒入硬化spacimen顶部。这黄色的Technovit 3040，防止来自管整个塑料块。 </li></ol>切片<ol><li>请用二甲苯Histoknife H（乙醇往往是不够好），并设置您的切片。角度约45 °的刀片。用二甲苯擦拭刀片前面舞台区，也因为这方面的清洁切片切片质量的关键。 </li><li>切管方和剥离，关闭使用的纸板刀的尖端。 <img src="/files/ftp_upload/old/188_2008_7_10_5/188_figure-02.jpg" alt="图2" width="818" height="243" /> </li><li>设置成夹头持有人的切片标本​​。 </li><li>一个载玻片上附加一个“按密封”绝缘体上硅，使安装节片室。用力按下绝缘体清洁台式幻灯片，从另一个侧面看，以确保该绝缘子是完全没有任何气泡附着。倒干净，温馨的DH 2 O（〜40℃）使用巴斯德吸管的幻灯片池，这里可以看出，水面变得平缓，没有凹或凸的形状的水平。 </li><li>开始节片。对于免疫的研究，我5毫米的节片。 原位杂交样品中 ，可以调整切片的厚度在〜3 - 8毫米的范围内，根据目的和信号强度。 </li><li>卡里的节片从刀片前面阶段的安装与水室，用一个小的画笔。一片水池上方握住画笔和降片敲钝尖清扫针水。片将扩大成圆形，尽快到达水面。 </li><li>当安装室内的水面面积大部分是充满节片，几乎一半的水使用巴斯德吸管吸。孵育幻灯片温暖过夜，以使样品完全干燥的幻灯片。 </li><li>反染色的DAPI（在TE为0.1mg/ml）1毫升五分钟。吸DAPI染色和干燥的样品。 </li><li>从幻灯片中删除的硅橡胶绝缘子。现在可以采取的样品图片。样品存放在4℃，避光。 </li></ol>

主要抗体对抗原或抗原表位标记：在PBT作出适当稀释。生物素标记的二抗：不同厂商的销售生物素标记的抗体。

plastic embedding and sectioning of xenopus laevis embryos

Watch this Scientific Journal Video about Plastic Embedding and Sectioning of Xenopus laevis Embryos at JoVE.com

Plastic Embedding and Sectioning of Xenopus laevis Embryos

塑料型材保持在薄薄的组织，可以用荧光抗体免疫染色的部分真正的组织形态，这种方法比较有用的，比许多类型的组织石蜡包埋或冰冻切片。染色，塑料包埋，切片的方法是在这个视频演示。

塑料嵌入和非洲爪蟾胚胎切片

plastic-embedding-and-sectioning-of-xenopus-laevis-embryos

Research

JoVE Journal

Biology

13.5K 次观看.  University of California, Irvine (UCI). 塑料型材保持在薄薄的组织，可以用荧光抗体免疫染色的部分真正的组织形态，这种方法比较有用的，比许多类型的组织石蜡包埋或冰冻切片。染色，塑料包埋，切片的方法是在这个视频演示。

视频: Plastic Embedding and Sectioning of Xenopus laevis Embryos

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

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

组织者和动物极植从非洲爪蟾胚胎和装配一个细胞粘附实验的剖析

科研

生物学

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.

从非洲爪蟾女性获得卵子

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.

制备及分馏爪蟾卵提取

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.

非洲爪蟾卵母细胞的微量注射

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.

组织测定动物第移植（ACT）的含量非洲爪蟾</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.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

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

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

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.

在面部移植<em&gt;非洲爪蟾</em&gt;胚胎

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

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

使用plusTipTracker软件测量微管动力学<em&gt;非洲爪蟾</em&gt;生长锥

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...

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.

非洲爪蟾作为一种模型来确定翻译减值

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