我们感谢与小鼠的繁殖和护理援助和实验室管理K.吉尔。也Muko​​yama实验室成员的技术帮助表示感谢。经费是由美国卫生Naitonal研究所院内研究计划。

1。收集小鼠胚胎肢体皮肤（E13.5〜E17.5） <ol><li>安乐死插入女性的批准程序。根据我们批准的动物协议，女性安乐死的CO 2曝光，然后由颈椎脱位保证。奠定其吸水纸巾上的动物和彻底浸泡在70％乙醇/ H 2 O从挤压瓶。 </li><li>解剖子宫的完整性和它放置在一个100 × 15毫米的Petri菜含有冰冷汉克斯“平衡盐溶液（HBSS）洗出的血液。 </li><li>分离和解剖胚胎。从胚胎中取出非常薄的羊膜。 </li><li> （选配）35 × 10毫米培养皿解剖一个单一的胚胎，如果每个胚胎需要进行基因分型。解剖尾巴被转移到0.2 ml的PCR基因分型管。 </li><li>切断胚胎的前肢和转让一环钳前肢成24个含有2毫升冰冷鲜在磷酸缓冲液（PBS）的4％多聚甲醛（PFA）以及板。 </li><li>修复与前肢在4 ° C过夜温柔Nutator搅拌机混合。 </li><li>翌日，删除的煤灰和温柔Nutator搅拌机混匀，室温5分钟2毫升的PBS清洗三次。 </li><li>转让100％的甲醇（甲醇）的前肢和储存：-20 ° C酶的冰箱（临界温度控制和不带自动除霜功能冰柜）。初级抗体上市100％甲醇处理后1个工作表。 </li><li>剥离过的皮肤用细镊子的前肢。患肢皮肤，脱水时，应该很容易从四肢分开。腹面朝上（掌侧）第一名的肢体。然后用细镊子，切开皮肤，如视频所示。下一步剖析围绕整个肢体，剥离皮肤无任何损害，轻轻地。 </li></ol> 2。全安装肢体皮肤的免疫组化染色<ol><li>于5ml聚丙烯圆底管肢体皮肤补充水分通过一系列甲醇/ PBT（PBS + 0.2％TRITON X - 100）（75％，50％，25％）每5分钟分级孵化。请注意，交换解决方案应小心，以避免损坏肢体皮肤。 </li><li>洗两次在PBT 5分钟，轻轻混匀，室温Nutator搅拌机。 </li><li>肢体皮肤座山羊血清/ PBS 0.2％TX100阻断山羊抗体的缓冲为10％或10％驴血清/ PBS 0.2％TX100封闭液驴二次抗体Nutator搅拌机上轻轻搅拌2小时室温。 </li><li>将800μl的初级抗体封闭液中（在表中列出的适当稀释）（35 × 10毫米的培养皿中，并转移到2毫升-离心管一个环钳肢体皮肤10％山羊血清/ PBS 0.2 TX100％或10％驴血清/ PBS 0.2％TX100）。肢体皮肤温和Nutator搅拌机混合，在4 ° C过夜孵育。请注意，可以同时使用多个来自不同的物种（如鼠单克隆抗体+多克隆抗体）的初级抗体。 </li><li>翌日，放入圆底5毫升聚丙烯管35 × 10毫米的培养皿和转让一环钳肢体皮肤与4毫升洗涤液（2％山羊血清/ PBS 0.2％TX100或2 ％驴血清/ PBS 0.2％TX100）。 </li><li>洗为15分钟，轻轻混匀，室温Nutator搅拌机五倍。 </li><li>放置在35 × 10毫米的培养皿中，并转移到2毫升-离心管一个环钳800μl封闭液中的抗体（10％山羊血清/ PBS 0.2％TX100或10％驴血清/肢体皮肤PBS 0.2％TX100）。通常情况下，我们使用从杰克逊ImmunoResearch（Cy3标记，Cy5的，1:300稀释）或Invitrogen公司的Alexa Fluor 488的Alexa Fluor 568的Alexa Fluor 633，1:250稀释的抗体。使用0.22μm的PVDF膜针头式过滤器，以消除二次抗体的聚合颗粒过滤器的二次抗体溶液。温柔Nutator搅拌机混匀，室温孵育1小时在黑暗中或用铝箔包裹的肢体皮肤。需要注意的是来自不同物种的不同荧光共轭抗体可以同时使用。 </li><li>放置在一个35 × 10毫米的培养皿中，并转移到5毫升聚丙烯圆底管一环钳与洗涤缓冲液（2％山羊血清/ PBS 0.2％TX100或2％驴血清/ PBS4毫升肢体皮肤0.2％TX100）。在黑暗中或用铝箔轻柔混匀，室温Nutator搅拌机包裹15分钟洗净五倍。 </li><li> （针对核counterstaining股权）孵育洗涤液（2％山羊血清/ PBS 0.2％TX100或2％驴血清/ PBS 0.2％TX100）为- PRO - 3（Invitrogen公司的T36054毫升​​肢体皮肤，1:3000稀释），在黑暗中或铝金属箔与温柔Nutator搅拌机搅拌10分钟，在室温temperatu包裹重。然后，洗5分钟三次4毫升在黑暗中或包裹铝箔与柔和的混合上的洗涤液（2％山羊血清/ PBS TX100或2％驴血清/ PBS 0.2％0.2％TX100）在室温Nutator搅拌机。需要注意的是强大的和具体的核染色观察到PRO - 3，在一个特定的排放量（氦氖633 nm激发）。 </li></ol> 3。安装肢体皮肤上滑动<ol><li>广场上一个35 × 10毫米培养皿的肢体皮肤。粉尘，水晶，纤维从皮肤内使用低照明体视显微镜下用细镊子，以避免大量的照片漂白层中删除。 </li><li>一环钳传输肢体皮肤粘连微观幻灯片。放置在幻灯片上（即对盖玻片）向上的内在卧层皮肤。扁平化的皮肤，仔细地用细镊子和删除结转洗衣机Kimwipe缓冲区。 </li><li>在防褪色的安装媒体安装无气泡。我们使用25“X25”盖玻片。固化一个平坦的表面上（例如在黑暗中，样品安装使用延长黄金试剂在室温下放置过夜在黑暗中观看前）。长期储存，密封盖玻片的幻灯片和储存在4 ° C。 </li></ol> 4。共聚焦显微镜<ol><li>设置适当的激光荧光团。我们用三个激光源，包括488nm（氩气的Alexa Fluor 488和GFP），561nm的DPSS的Alexa Fluor 568和Cy3标记和氦氖633纳米（的Alexa Fluor 633，Cy5和到临3）Leica TCS SP5的共聚焦显微镜。 </li><li>使用顺序扫描工具，以避免或减少串扰两倍或三倍染色样本在所有染料中，将在同一时间兴奋。在连续扫描模式，图像将被记录在一个顺序。 </li><li>更多关于激光激发荧光染料和一般的信息可能建立在“共聚焦显微镜的生物学家”希布斯（2004） </li></ol> 5。代表性的成果全安装三重标签的共聚焦immnofluorescence显微镜E15.5小鼠前肢在皮肤泛内皮细胞标记抗体的PECAM - 1（图1A - C，D蓝色），神经丝标记2H3（图1A绿色），平滑肌细胞标记αSMA（图1A，B红）显示模式的特点αSMA+动脉分支，与肢体皮肤2H3 +末梢神经保持一致。除了血管分支，这个四肢皮肤血管模型是用来研究淋巴管分支使用的抗体，淋巴管内皮细胞标志物LYVE - 1（图1D，E红色）和Neuropilin2（NRP2）（图1D，F绿图案）。 <img src="/files/ftp_upload/2620/2620fig1.jpg" alt="图1" /> 图1（AC）动脉配合在胚胎肢体皮肤的末梢神经。全安装三重标签的共聚焦immnofluorescence显微镜与PECAM - 1（交流，蓝）的神经丝蛋白标记2H3（绿色），和平滑肌细胞标记αSMA（A和B，红泛内皮细胞标记抗体）所示。在E15.5，2H3 +末梢神经（空心箭头）副动脉（箭头），αSMA覆盖+平滑肌细胞。 （DF）的淋巴管在胚胎肢体皮肤血管。三重标签的共聚焦immnofluorescence显微镜与泛内皮细胞标记抗体PECAM - 1（深蓝色），淋巴管内皮细胞标志物LYVE - 1（D和E，红）和Neuropilin2（NRP2）（D和F，绿色）所示。淋巴管是由两个LYVE - 1和NRP2可视化，而LYVE - 1是由巨噬细胞（开放箭头）的一个子集表达。比例尺：100μm的。

<ol>
	<li>Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+nerves+determine+the+pattern+of+arterial+differentiation+and+blood+vessel+branching+in+the+skin.">Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin.</a> Cell. 109, 693-705 (2002).</li><li>Mukouyama, Y. S., Gerber, H. P., Ferrara, N., Gu, C., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve-derived+VEGF+promotes+arterial+differentiation+via+neuropilin+1-mediated+positive+feedback.">Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback.</a> Development. 132, 941-952 (2005).</li><li>Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+distinction+and+angiogenic+interaction+between+embryonic+arteries+and+veins+revealed+by+ephrin-B2+and+its+receptor+Eph-B4.">Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4.</a> Cell. 93, 741-753 (1998).</li><li>Gerety, S. S., Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetrical+mutant+phenotypes+of+the+receptor+EphB4+and+its+specific+transmembrane+ligand+ephrin-B2+in+cardiovascular+development.">Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development.</a> Mol Cell. 4, 403-414 (1999).</li></ol>

血管系统是胚胎发育过程中以及在成人器官维护和生殖功能的器官发育的关键，因为它提供充足的氧气和养分的器官。适当的血管网，以及建立与血管生成的复杂和多步既存的毛细血管网，是具有高度支化结构和层次结构重组的过程。虽然许多作品已被证明在这些过程中涉及的各种分子，它没有明确如何机关或者其组件提供的信号，以促进血管发展的循序渐进的过程，包括内皮分化和血管分支的?...

抗体 泛内皮细胞标记物 <table border="1"><tbody><tr><td> 抗体 </td><td> 物种 </td><td> 公司 </td><td> 目录＃ </td><td> 工作条件 </td></tr><tr><td> PECAM - 1 </td><td>亚美尼亚仓鼠（男） </td><td> Chemicon公司</td><td> MAB1398Z </td><td> 1:100稀释＃1 </td></tr><tr><td> PECAM - 1 </td><td>鼠（M） </td><td>屋宇署</td><td> 553369 </td><td> 1:300稀释</td></tr><tr><td> VEGFR2 </td><td>鼠（M） </td><td> eBioscience公司</td><td> 14-5821-82 </td><td> 1:200稀释</td></tr><tr><td> CD34 + </td><td>鼠（M） </td><td> eBioscience公司</td><td> 13-0341 </td><td> 1:300稀释</td></tr><tr><td> IV型胶原</td><td>兔（P） </td><td> ABD Serotec </td><td> 2150年至1470年</td><td> 1:300稀释＃2 </td></tr></tbody></table> 动脉内皮细胞标记物 <table border="1"><tbody><tr><td> 抗体 </td><td> 物种 </td><td> 公司 </td><td> 目录＃ </td><td> 工作条件 </td></tr><tr><td> Neuropilin1 </td><td>兔（P） </td><td>亚历科洛德金的实验室＃3 </td><td></td><td> 1:3000稀释</td></tr><tr><td> Unc5H2 </td><td>山羊（P） </td><td>研发</td><td> AF1006 </td><td> 1:200稀释</td></tr></tbody></table> 静脉内皮细胞标记物 <table border="1"><tbody><tr><td> 抗体 </td><td> 物种 </td><td> 公司 </td><td> 目录＃ </td><td> 工作条件 </td></tr><tr><td> EphB4 </td><td>山羊（P） </td><td>研发</td><td> AF446 </td><td> 1:100稀释</td></tr></tbody></table> 淋巴管内皮细胞标记物 <table border="1"><tbody><tr><td> 抗体 </td><td> 物种 </td><td> 公司 </td><td> 目录＃ </td><td> 工作条件 </td></tr><tr><td> LYVE - 1＃4 </td><td>兔（P） </td><td> Abcam公司</td><td> ab14917 </td><td> 1:200稀释</td></tr><tr><td> LYVE - 1＃4 </td><td>鼠（M） </td><td> MBL </td><td> D225 - 3 </td><td> 1:200稀释</td></tr><tr><td>的Prox - 1 </td><td>兔（P） </td><td> Chemicon公司</td><td> AB5475 </td><td> 1:1000稀释</td></tr><tr><td>的Prox - 1 </td><td>山羊（P） </td><td>研发</td><td> AF2727 </td><td> 1:50稀释</td></tr><tr><td> Neuropilin2 </td><td>兔（P） </td><td>细胞信号转导</td><td> 3366 </td><td> 1 100稀释</td></tr><tr><td> podoplanin的</td><td>叙利亚仓鼠（M） </td><td>杂交瘤细胞银行</td><td> 8.1.1 </td><td> 1:200稀释</td></tr></tbody></table> 平滑肌细胞/周细胞标记 <table border="1"><tbody><tr><td> 抗体 </td><td> 物种 </td><td> 公司 </td><td> 目录＃ </td><td> 工作条件 </td></tr><tr><td> αSMA- CY3 </td><td>鼠标（M）＃5 </td><td>西格玛</td><td> C - 6198 </td><td> 1:500稀释＃6 </td></tr><tr><td> NG2 </td><td>兔（P） </td><td> Chemicon公司</td><td> AB5320 </td><td> 1:200稀释</td></tr><tr><td> SM22α </td><td>兔（P） </td><td> Abcam公司</td><td> ab14106 </td><td> 1:200稀释</td></tr></tbody></table> 用于绿色荧光蛋白记者的抗体<table border="1"><tbody><tr><td> 抗体 </td><td> 物种 </td><td> 公司 </td><td> 目录＃ </td><td> 工作条件 </td></tr><tr><td>绿色荧光蛋白</td><td>兔（P） </td><td> Invitrogen公司</td><td> A11122 </td><td> 1:300稀释</td></tr><tr><td>绿色荧光蛋白</td><td>鼠（M） </td><td> Nacalai tesque </td><td> 04404-84 </td><td> 1:1000稀释</td></tr><tr><td>绿色荧光蛋白</td><td>鸡（P） </td><td> Chemicon公司</td><td> P42212 </td><td> 1:300稀释</td></tr></tbody></table> LacZ报告的抗体<table border="1"><tbody><tr><td> 抗体 </td><td> 物种 </td><td> 公司 </td><td> 目录＃ </td><td> 工作条件 </td></tr><tr><td> β-半乳糖苷酶</td><td>兔（P） </td><td> MP生物医学</td><td> 55976 </td><td> 1:5000稀释</td></tr><tr><td> β-半乳糖苷酶</td><td>山羊（P） </td><td> ABD Serotec </td><td> 4600-1409 </td><td> 1:500稀释</td></tr><tr><td> β-半乳糖苷酶</td><td>鸡（P） </td><td> Abcam公司</td><td> ab9361 </td><td> 1:200稀释</td></tr></tbody></table> 周边轴突的抗体<table border="1"><tbody><tr><td> 抗体 </td><td> 物种 </td><td> 公司 </td><td> 目录＃ </td><td> 工作条件 </td></tr><tr><td> 2H3 </td><td>鼠标（M）＃7 </td><td>杂交瘤细胞银行</td><td> 2H3 </td><td> 1:200稀释</td></tr><tr><td> Tuj1 </td><td>鼠标（M）＃8 </td><td> Covance公司</td><td> MMS - 435P </td><td> 1:500稀释</td></tr><tr><td>外周</td><td>兔（P） </td><td> Chemicon公司</td><td> AB1530 </td><td> 1:1000稀释</td></tr></tbody></table> 迁移的雪旺氏细胞的抗体<table border="1"><tbody><tr><td> BFABP </td><td>兔（P） </td><td>托马斯米勒的实验室＃9 </td><td></td><td> 1:3000稀释</td></tr></tbody></table> （P）多克隆抗体（M）：单克隆抗体 ＃1：山羊抗亚美尼亚仓鼠Cy3标记（杰克逊ImmunoResearch 127-165-160）抗体，应作为辅助抗体。 ＃2：IV型胶原抗体可以用来检测血管原位杂交后。 ＃3：Neuropilin1抗体请亚历科洛德金的约翰霍普金斯大学的实验室提供。羊抗人Neuropilin1抗体是在R＆D（AF3870），虽然我们还没有测试它。 ＃4：LYVE - 1抗体检测胚胎皮肤中的巨噬细胞的一个子集。 ＃5：反αSMA抗体小鼠IgG2a单克隆抗体。 ＃6：Cy3标记共轭αSMA抗体一起孵育1小时在室温下用秒其他初级抗体的次级抗体。 ＃7：2H3抗体对神经丝鼠IgG1单克隆抗体。 ＃8：Tuj1抗体小鼠IgG2a对神经元特异性III类β-微管蛋白的单克隆抗体。 ＃9：BFABP（脑特异性脂肪酸结合蛋白）抗体是好心的Thomas Müller的马克斯 - 德尔布吕克中心分子医学实验室提供。

whole-mount immunohistochemical analysis for embryonic limb skin vasculature: a model system to study vascular branching morphogenesis in embryo

全安装整个血管成像的免疫组织化学分析的关键是了解分支形态发生的细胞机制。我们已经开发出患肢皮肤血管的模型来研究血管发展，其中一个预先存在的原始毛细血管丛是一个层次的分支血管网络重组。全安装多个标签的共聚焦显微镜允许以及完整的血管内皮细胞，周细胞和平滑肌细胞的细胞成分，包括使用特定的荧光标记，强大的成像。这个患肢皮肤血管模型与遗传研究进展，更好地了解血管的发展和图案的分子机制。患肢皮肤血管模型已被用于研究的分化和图案动脉末梢神经如何提供一个空间模板。此视频文章介绍了一个简单而强大的协议染色完整的血管与血管特异性抗体和荧光抗体，这是适用于我们能够遵循的血管发育过程中的吻合​​血管的胚胎器官。

Watch this Scientific Journal Video about Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo at JoVE.com

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

我们介绍整个安装的免疫组织化学和激光扫描共聚焦显微镜分析复杂的小鼠胚胎肢体皮肤血管网的形成多个标签。

全安装胚胎肢体皮肤血管的免疫组织化学分析：一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

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Biology

17.5K 次观看.  National Heart, Lung, and Blood Institute, National Institutes of Health. 我们介绍整个安装的免疫组织化学和激光扫描共聚焦显微镜分析复杂的小鼠胚胎肢体皮肤血管网的形成多个标签。

视频: Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

整装在鸡的抗体染色方法

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

小鼠胚胎的龙文化，一个系统来评价分枝的分子机制

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em在原位</em&gt;改编为海洋软骨鱼类杂交整装胚胎发育和细胞组织学

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

鼠胚体外发育无血清全胚胎培养系统

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...