Name: techniques for rapidly sampling six crucial organs in adult xenopus
Uploaded: 2024-02-16T15:00:00.000+00:00
Duration: 5 min 10 s
Description: Watch this Scientific Journal Video about Author Spotlight: Standardizing Tissue Sampling in Proteomics and Immunochemistry Research at JoVE.com

这项工作得到了 NIH 的 OD 拨款R24OD031956的支持。我们感谢 Samantha Jalbert、Jill Ralston 和 Cora Anderson 的帮助和支持，以及我们的编辑和匿名同行评审员的有用反馈

所有实验均按照哈佛医学院IACUC（机构动物护理和使用委员会）（IS 00001365_3）的规则和规定进行。灌注和未灌注的成熟白化雄性 非洲爪蟾（Xenopus laevis）的代表性结果均显示。
1. 实验准备 
注意:如果在采样前遵循灌注方案8 ，请跳至步骤 2.2。
<ol><li>确保研究机构已批准本协议中描述的安乐死技术。</li>
	<li>制备 5 g/L MS-222（甲基磺酸三卡因）和 5 g/L 碳酸氢钠的溶液（参见 材料表）。该体积必须大于完全覆盖被安乐死的动物所需的体积。检查 pH 值以确保其为 ≥7。</li>
	<li>通过将 非洲爪蟾 放入安乐死溶液中进行初级安乐死;动物将保持在水中总共 1 小时。</li>
	<li>设置解剖站，以便在取样后立即在冷冻 PBS 或 0.7x PBS9 （取决于实验需要）中冲洗所有组织，检查并在 5x（或更大）放大灯下修剪。该站还必须使用户能够更换所有镊子和剪刀，或者在两次使用之间将它们擦拭干净。</li>
	<li>一旦青蛙在溶液中放置 1 小时，初级安乐死就完成了。取出青蛙并通过执行脚捏来检查疼痛反应的丧失。</li>
	<li>记录动物的适当详细信息，例如物种、品系、性别、年龄和健康状况，以及是否灌注过。称量 非洲爪蟾 的重量并进行任何其他测量，例如鼻子通风口长度。</li>
	<li>将青蛙放在背上，将四肢固定在身体附近（图1）。</li>
	<li>使用解剖剪刀，切开皮肤，沿着中线，然后横向切成两个皮瓣。</li>
	<li>参 照图 2，识别白线并用镊子抓住它并将其从腔腔中拉开。用剪刀小心地切开肌肉组织。从腔壁上做两个襟翼。将所有襟翼切开或固定在一边。</li>
	<li>确定仍在跳动的心脏。使用解剖剪刀减少喙突骨（图2），以更好地进入心脏。 注意:如果心脏在采样前停止跳动，则应注意样本新鲜度已受到影响。</li>
</ol>2. 抽样
注意:如果动物已经灌注，请跳至步骤 2.2。
<ol><li>识别薄心包并用组织钳将其拉紧（图3）。</li>
	<li>使用虹膜切除术剪刀的尖端，轻轻地穿孔心包，注意不要割伤下面的组织。将心包从心脏的 3 个腔室中剥离出来。</li>
	<li>使用镊子抓住心室的顶点，确定它附着在耳廓和动脉干上的位置（图4），并将其切入这些附着物下方（图5）。如果需要，修剪心室，使耳廓或动脉干的组织不可见，并且心室内仍可见浅色瓣膜组织。 注意:在未灌注的动物中，切除心室可能符合继发性安乐死的条件。</li>
	<li>肝脏的 3 个叶将是可见的（图 6 和 图 7）。抓住左叶的边缘（在观察者的右侧）并轻轻提起它，使肝管和囊管可见（图8）。对这些附件下方波瓣的底部 1/3 进行采样（图 9）。</li>
	<li>为了更好地接触雌性青蛙的组织，切除卵巢是有帮助的。识别包裹在称为生发上皮的内脏腹膜层中的卵巢。轻轻移动波瓣，直到它们位于各自的一侧，使附着区域可见（图 10）。这些附着物直接位于配对肾脏的腹侧。</li>
	<li>使用剪刀，尽可能靠近肾脏切除卵巢，而不会损坏它们（图11）。</li>
	<li>检查肝脏的内侧叶（也称为前叶），并注意它如何通过肠系膜和肝胰管（也称为胆总管）连接到胃和十二指肠（图 6、 图 7 和 图 8）。</li>
	<li>使用虹膜切除术剪刀切断肠系膜、肝十二指肠韧带以及与十二指肠相接的肝胰管。切断胰腺和肝胰管与肝脏内侧叶的连接，以免附着深色肝组织（图12）。</li>
	<li>用齿镊子夹住胃，用组织镊子夹住胰腺的上端。在5倍放大镜下，轻轻地将胰腺从胃中取出（图13）。 注意:如果它没有干净地脱落，剩余的胰腺组织将是可见的，并且可以被切成碎片。或者，可以使用虹膜切除术剪刀和组织镊子有方法地分离胰腺。</li>
	<li>参考 图 14A，识别膀胱并将其切除，尽可能靠近泄殖腔切开。丢弃此纸巾。</li>
	<li>参考 图 14B，识别大肠并将其拉紧，以尽可能靠近泄殖腔切断大肠。切除并丢弃整个消化道，切断腹膜与脾脏相连的地方。现在，肥胖的身体将完全可以接触到。</li>
	<li>挑逗脂肪体，使它们位于各自的两侧。脂肪体与腹膜相连的肾脏上方区域将是可见的。抓住左侧脂肪体的底部（观察者的右侧），并用剪刀将其从腹膜上剪下，留出一小块边缘，以免肾脏受损（图15）。</li>
	<li>取出并丢弃剩余的脂肪体。配对的肾脏现在将完全可见。</li>
	<li>在雌性青蛙或具有明显退化输卵管的雄性青蛙中，抓住输卵管并将其从肾脏和泄殖腔中拉开（图16）。切开输卵管与泄殖腔相接的地方，继续将其从肾脏拉开，当它们变得明显时，切断任何明显的腹膜附着物。丢弃此纸巾。</li>
	<li>对剩余的输卵管重复此过程。</li>
	<li>肾脏仍然覆盖着透明的腹膜（腹膜后）10。使用镊子抓住肾脏并切断其下端的腹膜。</li>
	<li>将肾脏从腔腔中取出，使用剪刀将腹膜尽可能靠近肾脏切开，而不会损坏它们（图17）。</li>
	<li>在 5 倍放大镜下，切去多余的腹膜和任何其他剩余组织（脂肪体、脾脏）。如果青蛙是雌性，请确保切除任何剩余的卵巢组织（图18）。如果青蛙是雄性，小心地切除睾丸并检查是否有退化的输卵管，如果不放大，可能无法看到（图19）。</li>
	<li>从动物身上取下别针，将其翻转到腹腔，然后重新固定动物的四肢。</li>
	<li>选择任一后肢进行采样，并固定该肢体的脚。</li>
	<li>从腓肠肌/胫腓骨上方取下杏仁形的皮瓣（图20）。</li>
</ol>

成年 非洲爪蟾 器官的标准化解剖和采样方案

<ol>
	<li>Porro, L. B., Richards, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+dissection+of+the+model+organism+Xenopus+laevis+using+contrast-enhanced+computed+tomography.">Digital dissection of the model organism Xenopus laevis using contrast-enhanced computed tomography.</a> J Anat. 231 (2), 169-191 (2017).</li><li>Ruehl-Fehlert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+guides+for+organ+sampling+and+trimming+in+rats+and+mice--part+1.">Revised guides for organ sampling and trimming in rats and mice--part 1.</a> Exp Toxicol Pathol. 55 (23), 91-106 (2003).</li><li>Kittel, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+guides+for+organ+sampling+and+trimming+in+rats+and+mice--Part+2.+A+joint+publication+of+the+RITA+and+NACAD+groups.">Revised guides for organ sampling and trimming in rats and mice--Part 2. A joint publication of the RITA and NACAD groups.</a> Exp Toxicol Pathol. 55, 413-431 (2004).</li><li>Morawietz, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+guides+for+organ+sampling+and+trimming+in+rats+and+mice+-+Part+3+-+A+joint+publication+of+the+RITA+and+NACAD+groups.">Revised guides for organ sampling and trimming in rats and mice - Part 3 - A joint publication of the RITA and NACAD groups.</a> Exp Toxicol Pathol. 55, 433-449 (2004).</li><li>Patmann, M. D., Shewade, L. H., Schneider, K. A., Buchholz, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+tadpole+tissue+harvest.">Xenopus tadpole tissue harvest.</a> Cold Spring Harb Protoc. 2017 (11), 097675 (2017).</li><li>L&#337;w, P., Moln&#225;r, K., Kriska, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+a+Frog+(Rana+sp.).">Dissection of a Frog (Rana sp.).</a> Atlas of Animal Anatomy and Histology. , 213-263 (2016).</li><li>O'Rourke, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphibians+used+in+research+and+teaching.">Amphibians used in research and teaching.</a> ILAR J. 48 (3), 183-187 (2007).</li><li>Jonas-Closs, R. A., Peshkin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+rapid+blood+perfusion+in+Xenopus.">Effective rapid blood perfusion in Xenopus.</a> JoVE. (issue), e65287 (2023).</li><li>Balls, M., Worley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphibian+cells+in+vitro.+II.+Effects+of+variations+in+medium+osmolarity+on+a+permanent+cells+line+isolated+from+Xenopus.">Amphibian cells in vitro. II. Effects of variations in medium osmolarity on a permanent cells line isolated from Xenopus.</a> Exp Cell Res. 76 (2), 333-336 (1973).</li><li>Holz, P. H., Raidal, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+renal+anatomy+of+exotic+species.">Comparative renal anatomy of exotic species.</a> Vet North Am Exot Anim Pract. 9 (1), 1-11 (2006).</li><li>Trott, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+Mycobacterium+ulcerans-like+infection+in+a+colony+of+African+tropical+clawed+frogs+(Xenopus+tropicalis).">Characterization of a Mycobacterium ulcerans-like infection in a colony of African tropical clawed frogs (Xenopus tropicalis).</a> Comp Med. 54 (3), 309-317 (2004).</li><li>Fremont-Rahl, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+liflandii+outbreak+in+a+research+colony+of+Xenopus+(Silurana)+tropicalis+frogs.">Mycobacterium liflandii outbreak in a research colony of Xenopus (Silurana) tropicalis frogs.</a> Vet Pathol. 48 (4), 856-867 (2011).</li><li>Green, S. L., Parker, J., Davis, C., Bouley, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+hyperstimulation+syndrome+in+gonadotropin-treated+laboratory+South+African+clawed+frogs+(Xenopus+laevis).">Ovarian hyperstimulation syndrome in gonadotropin-treated laboratory South African clawed frogs (Xenopus laevis).</a> J Am Assoc Lab Anim Sci. 46 (3), 64-67 (2007).</li><li>Vitt, L. J., Caldwell, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+amphibians+and+reptiles.+Herpetol.">Anatomy of amphibians and reptiles. Herpetol.</a> Herpetol. , 35-81 (2009).</li></ol>

提交人声明没有竞争利益。

由于该协议旨在最大限度地提高新鲜度，因此某些样品可能包含不需要的组织。例如，肝胰管和一些肠系膜与胰腺一起取样，一些腹膜组织、肾上腺和输尿管将始终与配对的肾脏一起取样。 如果新鲜度不是问题，那么可以使用改进的技术实现更精确的采样。
器官的外观和位置在非洲 爪蟾的性别和物种之间具有可比性。然而，根据动物是否灌注过，组织的颜色会有很大...

尽管成年非洲爪蟾的"数字解剖"已经可用1，但如果没有其他成年模型（例如小鼠2,3,4）的详细指导，成年非洲爪蟾的可复制器官和组织采样仍然具有挑战性。本文旨在为成年非洲爪蟾的准确和可复制的器官取样提供明确的指导，类似于目前可用于其幼虫的器官取样5.强调易于完成，以保持最大的新鲜度，并使所有用户都可以访问该协议。
虽然有针对 Rana sp.6 的详尽解剖指南，以及针对其他anurans 7 的大量课堂解剖指南，但目前没有可用的非洲爪蟾解剖和取样指南。对于那些不熟悉采样实践或两栖动物解剖学的人来说，非洲爪蟾和其他无蟾之间的微小差异使得这些资源对于可复制的组织取样来说并不理想。
许多有价值的组织不包括在本指南中，甚至被丢弃;这是为了保证组织的新鲜度。六个样本足够有限，以确保无论使用者的经验或技能水平如何，都可以在心脏开始跳动后一小时内收集到这些组织。关于收集许多其他组织的更先进和详细的指南正在作为单独的配套文件编写中。
对于经验不足的用户，始终建议首先在因实验以外的原因被安乐死的动物上尝试此协议，然后再对任何难以替代的动物（即转基因、高龄动物等）进行采样。理想情况下，所有取样的动物都是健康的，如果是雌性，则在过去两周内不会排卵。

<table><tbody><tr><td>5x Magnifying Glass with LED Light and Stand</td><td>amazon.com</td><td>B08QJ6J8P1</td><td>light must not produce heat</td></tr><tr><td>Disposable Transfer Pipets</td><td>VWR</td><td>414004-036</td><td></td></tr><tr><td>Dissecting Fine-Pointed Forceps</td><td>Fisher Scinetific</td><td>08-875</td><td></td></tr><tr><td>Dissecting scissors sharp piont, straight 6.5&quot;</td><td>VWR</td><td>76457-374</td><td></td></tr><tr><td>Dissection Tray</td><td>Fisher Scinetific</td><td>14-370-284</td><td>styrofoam sheets are an acceptable alternative</td></tr><tr><td>Euthanasia container</td><td>US Plastic&amp;nbsp;</td><td>Item&amp;nbsp;2860</td><td>alternative opaque containers acceptable</td></tr><tr><td>Euthanasia container lid</td><td>US Plastic&amp;nbsp;</td><td>Item&amp;nbsp;3047</td><td></td></tr><tr><td>Iridectomy Scissors 6&quot;</td><td>vwr</td><td>470018-938</td><td>iris scissors are an acceptable alternative</td></tr><tr><td>MS-222: Syncaine (formerly tricaine)</td><td>Pentair AES</td><td>TRS1</td><td></td></tr><tr><td>PBS 1x</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Sodium Bicarbonate, Powder, USP</td><td>Fisher Scientific</td><td>18-606-333</td><td></td></tr><tr><td>Specimen Forceps, Serrated</td><td>VWR</td><td>82027-442</td><td></td></tr><tr><td>T-Pins for Dissecting</td><td>Fisher Scinetific</td><td>S99385</td><td></td></tr></tbody></table>

techniques for rapidly sampling six crucial organs in adult xenopus

一百多年来，非洲爪蟾一直是理解脊椎动物发育和疾病的强大模式生物。虽然胚胎的实验分析和解剖技术已经有充分的记录，但对成年非洲爪蟾结构和器官的描述，以及与成人一起工作的技术，尚未得到更新，以考虑到定量蛋白质组学和单细胞转录组学等现代方法的要求。从细胞类型和基因为中心的视角，需要在胚胎阶段与在成体组织中进行对比观察。在幼虫向成虫的过渡过程中，幼虫器官的整体结构、形态和解剖位置都发生了重大变化，最明显的是在大规模重塑过程中。建立健全的器官鉴定和解剖标准对于确保在不同实验室进行的研究得出的数据集能够保持一致至关重要。本方案确定了成年非洲爪蟾的六个器官，展示了对成年非洲爪蟾的心室、肝脏、脂肪体、胰腺、配对肾脏和皮肤的解剖和取样方法。根据保存方法的不同，解剖器官可用于定量蛋白质组学、单细胞/细胞核转录组学、原位杂交、免疫组化、组织学等。该协议旨在标准化组织取样并促进成人器官系统的多实验室研究。

通过使用 图 1 至 图 20 并遵循该协议的所有步骤，心室、肝左叶、胰腺、左脂肪体、成对的肾脏和皮瓣在安乐死后一小时内净地切除。在这段时间内，对样品进行冲洗和修整，使其出现，如图 21 所示。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66489/66489fig1.JPG"> 图 1:...

Watch this Scientific Journal Video about Author Spotlight: Standardizing Tissue Sampling in Proteomics and Immunochemistry Research at JoVE.com

作者聚焦:蛋白质组学和免疫化学研究中的组织采样标准化

本文介绍了在成年 非洲爪蟾 中可以快速轻松地访问的六个重要且多样化的器官样本指南:心室、肝叶、胰腺、脂肪体、成对的肾脏和皮肤。

成年非洲爪蟾六个关键器官快速取样技术

Xenopus has been a powerful model organism for understanding vertebrate development and disease for over a hundred years. While experimental analysis and ...

在这项工作之前，我开发了一种用于快速两栖动物灌注的方案。在取样进行蛋白质组学和免疫化学等研究之前，应使用该协议，在这些研究中，组织中存在过多的血液是不可取的。尽管有许多图表和其他辅助工具可用，但目前还没有关于如何快速可靠地从成年非洲爪蟾中解剖组织的综合指南。我们希望拥有标准化的采样技术将使实验室能够轻松共享组织。在由于生物安全问题或运输限制而无法进出口动物的情况下，这尤其有利。

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Techniques for Rapidly Sampling Six Crucial Organs in Adult Xenopus

838 次观看.  Harvard Medical School. 在这项工作之前，我开发了一种用于快速两栖动物灌注的方案。在取样进行蛋白质组学和免疫化学等研究之前，应使用该协议，在这些研究中，组织中存在过多的血液是不可取的。尽管有许多图表和其他辅助工具可用，但目前还没有关于如何快速可靠地从成年非洲爪蟾中解剖组织的综合指南。我们希望拥有标准化的采样技术将使实验室能够轻松共享组织。在由于生物安?...

视频: 作者聚焦:蛋白质组学和免疫化学研究中的组织采样标准化

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

干细胞样<em&gt;爪</em&gt;胚胎外植体，研究早期神经发育特点<em&gt;体外</em&gt;和<em&gt;在体内</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

科研

发育生物学

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

活蛙 (爪蟾) 胚胎单细胞代谢的微探针毛细管电泳质谱

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

化学

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

同时前和突触后电生理记录<em&gt;爪蟾</em神经肌肉文化

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

神经科学

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

评估主要神经再生<em&gt;爪</em&gt;胚胎使用免疫染色

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

活的嗅觉途径的功能评价

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro and in vivo. Fluorescent probes are particularly useful in determining the bio-distribution of administered large molecules, where the addition of a small-molecule fluorescent label is unlikely to affect the kinetics or bio-distribution of the compound. A variety of methods exist to examine bio-distribution that vary significantly in the amount of effort required and whether the resulting measurements are fully quantitative, but using multiple methods in conjunction can provide a rapid and effective system for analyzing bio-distributions.
Ex vivo whole-organ imaging is a method that can be used to quickly compare the relative concentrations of fluorescent molecules within tissues and between multiple types of tissues or treatment groups. Using an imaging platform designed for live-animal or whole-organ imaging, fluorescence within intact tissues can be determined without further processing, saving time and labor while providing an accurate picture of the overall bio-distribution. This process is ideal in experiments attempting to determine the tissue specificity of a compound or for the comparison of multiple different compounds. Quantitative tissue histology on the other hand requires extensive further processing of tissues in order to create a quantitative measure of the labeled compounds. To accurately assess bio-distribution, all tissues of interest must be sliced, scanned, and analyzed relative to standard curves in order to make comparisons between tissues or groups. Quantitative tissue histology is the gold standard for determining absolute compound concentrations within tissues.
Here, we describe how both methods can be used together effectively to assess the ability of different administration methods and compound modifications to target and deliver fluorescently labeled molecules to the central nervous system1.

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Ex vivo whole-organ imaging is a rapid method for determining the relative concentrations of fluorescently labeled compounds within and between tissues or treatment groups. Conversely, quantitative fluorescence histology, while more labor intensive, allows for the quantification of the absolute tissue levels of labeled molecules.

指某东西的用途<em&gt;离体</em&gt;全器官成像和定量组织组织学确定荧光标记分子的生物分布

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...

生物学

An Apical Resection Model in the Adult Xenopus tropicalis Heart

It is known that in adult mammals, the heart has lost its regenerative capacity, making heart failure one of the leading causes of death worldwide. Previous research has demonstrated the regenerative ability of the heart of the adult Xenopus tropicalis, an anuran amphibian with a diploid genome and a close evolutionary relationship with mammals. Additionally, studies have shown that following ventricular apex resection, the heart can regenerate without scarring in X. tropicalis. Consequently, these previous results suggest that X. tropicalis is an appropriate alternative vertebrate model for the study of adult heart regeneration. A surgical model of cardiac regeneration in the adult X. tropicalis is presented herein. Briefly, the frogs were anesthetized and fixed; then, a small incision was made with iridectomy scissors, penetrating the skin and pericardium. Gentle pressure was applied to the ventricle, and the apex of the ventricle was then cut out with scissors. Cardiac injury and regeneration were confirmed by histology at 7-30 days post resection (dpr). This protocol established an apical resection model in adult X. tropicalis, which&#160;can be employed to elucidate the mechanisms of adult heart regeneration.

An Apical Resection Model in the Adult Xenopus tropicalis Heart

Xenopus tropicalis is an ideal model for regenerative research as many of its organs possess a remarkable regenerative capacity. Here, we present a method for constructing a heart injury model in X. tropicalis via apex resection.

热带非洲爪蟾成人心脏的根尖切除模型

It is known that in adult mammals, the heart has lost its regenerative capacity, making heart failure one of the leading causes of death worldwide. ...

医学

用于转录组学和蛋白质组学的非洲爪蛙血液灌注

Effective Rapid Blood Perfusion in Xenopus

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol in Xenopus, aimed at a consistent and drastic reduction of blood within all tissues, is defined. Perfusion is carried out by inserting a needle directly into the ventricle of the heart and pumping heparinized phosphate-buffered saline (PBS) through the vascular system. The procedure can be completed in approximately 10 min per animal. The blood is dominated by a few highly abundant proteins and cell types, creating numerous issues as these proteins mask most other molecules and cell types of interest. The reproducible characterization of adult Xenopus tissues with quantitative proteomics and single-cell transcriptomics will benefit from applying this protocol prior to organ sampling. The protocols for tissue sampling are defined in companion papers. These procedures are aimed at the standardization of practices across Xenopus of different sex, age, and health status, specifically X. laevis and X. tropicalis.

作者聚焦：非洲爪蟾中的有效快速血液灌注  

Presented here is an effective rapid blood perfusion protocol to prepare tissue samples from African clawed frogs for transcriptomics and proteomics studies.

非洲爪蟾的有效快速血液灌注

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol ...

成年非洲爪蟾六个关键器官快速取样技术

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此视频中的章节

干细胞样

技术目标显微注射到显影

胚胎血管生成的可视化和定量分析

活蛙 (爪蟾) 胚胎单细胞代谢的微探针毛细管电泳质谱

同时前和突触后电生理记录

评估主要神经再生

活的嗅觉途径的功能评价

指某东西的用途

热带非洲爪蟾成人心脏的根尖切除模型

非洲爪蟾的有效快速血液灌注