我们感谢克里斯托夫·恩格勒特教授和Didier Stainier教授送礼的的Tg（wt1b:GFP）LI1和TG（kdrl:EGFP）S843 株，分别与台湾斑马鱼核心设施提供的Tg（kdrl:mCherry）CI5。这项研究是由赠款科技（96-2628-B-029-002-MY3，101-2313-B-029-001，102-2628-B-029-002-MY3台湾省，支持102- 2321-B-400-018）。

伦理声明:对斑马鱼的所有试验程序都经东海大学的机构动物护理和使用委员会（IRB批准文号101-12），并按照批准的指导方针进行。 1.3β-HSD酶的活性染色股票方案<ol><li>制备反式dehydroandrosterone [10毫克/毫升的二甲亚砜（DMSO）。 </li><li>制备β-烟酰胺腺嘌呤二核苷酸水合物（1.2毫克/毫升的0.1M磷酸盐缓冲液，pH 7.2）。 </li><li>准备烟酰胺（维生素B3，50毫克/毫升H 2 O）。 </li><li>制备4-硝基蓝四唑（在70％的二甲基甲酰胺50毫克/毫升）。 </li><li>分装所有这些组件到离心管并储存在-20°C。 注意:根据我们的经验，反复冷冻和所有上述解决方案的等分试样解冻不影响3β-HSD活性染色的强度。 </li></ol> 2.W¯¯孔贴装3β-HSD活性染色注:3β-HSD的酶活性是在斑马鱼胚胎检测早28个小时后受精（HPF）培养时在28.5℃下，它是与HSD3B1 2,7- mRNA表达的开始相一致。在这项研究中整个安装3β-HSD活性染色协议是基于一个先前描述方法8和被确定为成功的显影斑马鱼7天受精后9。用于分析interrenal类固醇生成组织的血管的微环境，整个安装3β-HSD活性染色必须应用到转基因株系表达特定内皮荧光，例如为Tg（kdrl:EGFP）S843 14和TG（kdrl:mCherry）CI5 15 。为了分析interrenal类固醇生成组织考虑斑马鱼肾脏发育，3β-HSD活性染色可APPLI编到的Tg（wt1b:GFP）LI1鱼16，其中肾小球和pronephric小管的形成划定。 <ol><li>在鸡蛋水0.03％苯基硫脲培养他们（在去离子水0.06毫克/升礁盐）治疗发展斑马鱼，从12和24之间HPF一个阶段开始，抑制色素形成。 </li><li>在补充有0.1％吐温20（PFAT）在室温下（RT）或（B）2％PFAT 1小时过夜磷酸盐缓冲盐水（PBS）固定在（A）中的4％多聚甲醛（PFA）dechorionated胚胎或幼虫4℃或在4℃下2小时，接着在RT 4小时。 注意:PFA是通过吸入和皮肤接触有毒。它是破坏性的皮肤，粘膜，眼睛和上呼吸道。 </li><li>洗胚胎4倍10分钟，用PBS补充有在RT 0.1％吐温20（PBST）。 注意:这是在改变洗涤溶液不干燥样品的关键。 </li><li>刚准备站反应之前进不去溶液（ 表1）。准备，旋涡，并在不同的离心管A和B部分。 注意:添加所有组件直接到一个单管会引起强降水。 </li><li> A部分的整个解决方案添加到B部分的整个解决方案，拌匀准备染色液，立即分发1毫升到含有固定的，洗的胚胎各离心管。 </li><li>包裹离心管用铝箔保护他们免受光和地方在任一个旋转或轻微振荡在RT摇摆平台。 </li><li>凭经验确定通过显微镜监测显色信号的反应时间。轻微降水将随着时间的发展;但是，它们将不与反应过程产生干扰。 注:对于胚胎固定在28 HPF，染色在25℃下4小时产生在interrenal组织的明确信号。 </li><li>停止洗涤4倍于在PBST 10分钟的反应。一世f被不需要进一步免疫组织化学（IHC）分析，后固定染色的胚胎，用4％PFAT在RT 60分钟。否则，在4℃下保存的胚胎冲洗，直到进一步使用。 </li><li>对于全安装显微镜，清除染色，后固定，洗胚胎在PBS 50％甘油，投影片上朝上背侧定位它们，并使用在明亮的视场照明正置显微镜观察。 注意:为了使interrenal组织形态的高分辨率图像，平面安装3β-HSD活性染色和deyolked胚胎的分析，建议。由于interrenal类固醇合成组织定位正确的卵黄囊上述情况，interrenal组织的腹平贴分析允许interrenal形态17的直接和清晰的观察。 </li></ol> 3.3β-HSD活性染色胚胎IHC分析注:为了分析steroi的血管微环境dogenic组织，用转基因特异性内皮荧光报道的3β-HSD活性染色的胚胎经受横向vibratome切片和随后的免疫组织化学分析12,18。 <ol><li>后固定<ol><li>在4℃下后固定3β-HSD活性染色和洗涤胚胎用2％PFA补充有1％的Triton X-100 1小时并洗4倍于含有1％Triton X-100的10分钟的PBS（PBSTx）在室温。 </li></ol></li><li>嵌入<ol><li>制备4％的在PBS琼脂糖溶解在95℃下，分装成微量离心管，并存储在4-8℃的琼脂糖低熔点。 </li><li>熔体4％低熔点琼脂糖的等分试样在70℃下进行10分钟，然后保持等分试样在47℃。嵌入每个胚在熔融4％琼脂糖低熔点在用作一个模具1.5 ml离心管的分离的盖子，并允许冷却，直到固体。 </li></ol></li><li> Vibratome切片<ol><li>坚持一纸胶带到干p在vibratome的latform。 </li><li>由一个刀片除去从模具硬化琼脂糖块。应用强力胶到平台上的纸带，并琼脂糖块固定到所述纸带，具有朝上的样品的前侧。修剪琼脂糖块为梯形棱镜的形状，分别与大约4毫米，在上部和下部小面的每个侧8毫米。 </li><li>通过使用滴管冲洗用冷PBS将含有样品的琼脂糖块补​​充有0.5％的Triton X-100（0.5％PBSTx）。 </li><li>根据vibratome的使用说明书切片琼脂糖块成100微米的部分。不断冲洗琼脂糖块，防止干燥。由于每片被切断，用＆G注射器针头从vibratome删除它，转移到冷含0.5％PBSTx（500微升/孔）现货板。 </li><li>通过解剖显微镜下检查收集3β-HSD活性阳性的组织切片，并通过PI传送节佩特前端以其端截止（约1mm的开口内径）到含有冷0.5％PBSTx（150微升/孔）在96孔板中。的组织样品通常从该步骤后的琼脂糖块解离。 </li></ol></li><li>通透性和洗<ol><li>洗片6倍于室温在0.5％PBSTx 15分钟，在PBS中10分钟，和4倍于在PBS中10分钟补充有1％牛血清白蛋白/ 1％DMSO / 0.1％的Triton X-100（PBDTx）。 </li></ol></li><li>闭塞<ol><li>孵育在PBDTx 1小时2％胎牛血清（FCS）在RT切片。 </li></ol></li><li>主要抗体反应<ol><li>孵育切片1.5天在PBDTx含有第一抗体（ 例如 ，兔多克隆抗人纤连蛋白和小鼠抗人粘着斑激酶）在4℃下。 </li></ol></li><li>洗涤<ol><li>洗净切片6倍在RT在PBDTx 10分钟。 </li></ol></li><li>闭塞<ol><li>孵育1小时的切片在PB 2％FCS的DTx中在室温。 </li></ol></li><li>二级抗体反应<ol><li>在4℃下孵育1.5天在PBDTx含有二级抗体（荧光团缀合的山羊抗兔或抗小鼠IgG）的片。 </li></ol></li><li>洗涤<ol><li>洗片6倍于RT在PBDTx 10分钟和4倍，在室温在PBST 10分钟。 注:对于共焦显微镜分析，用PBS中的50％甘油清除样品。 </li></ol></li></ol> 4.共聚焦分析<ol><li>观察整个安装胚胎，并配备了10X / 0.5和20X / 0.75的物镜共聚焦显微镜的部分。 </li><li>对于荧光和3β-HSD染色同时检测，设置如下多轨模式:488nm的氩激光和检测GFP的505-530纳米带通滤波器; 543纳米的氦氖激光器和用于检测mCherry的560-615纳米的带通滤波器;并传输用于检测3β-HSD染色的光信道。针孔的大小是1艾雷单元，分辨率为1024×1024像素。 </li><li>组装的Z堆叠（具有6微米的间隔），为3D投影，并合并突起和明场，通过使用内置的共聚焦软件。 </li></ol>

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The authors have no competing financial interests to declare.

的3β-HSD活性的信号强度增加了反应过程。 4小时从28 HPF向前阶段反应后，检测所述3β-HSD活性的明确信号。然而，反应的持续时间，需要经验确定，这取决于测定的目的。在染色要求夜间处理的情况下，略微偏蓝的背景往往对样品开发。这个问题可以通过3β-HSD活性染色之前增加注视时间（ 例如 ，在在RT 4％PFAT 4小时）来克服。相比之下，overfixation，如固定过夜与RT 4％PFAT，导致了一个非常清晰的背景。然而，较长的时间就需要获取的信号的希望的强度。我们注意到，3β-HSD活性染色之前过夜，用2％，而不是4％PFAT定影样品通常产生在随后的荧光报告观察或我更理想的结果HC分析。然而，4％PFAT固定胚胎允许3β-HSD活性（1天之内完成）的快速检测，并会因此遗传或化学屏幕瞄准interrenal类固醇组织受益。 3β-HSD活性染色后后固定胚胎是必不可少的，因为化学成分的微量往往即使经过充分洗涤染色样品中坚持并会导致长期存放时间后高的背景。然而，如果3β-HSD活性染色胚要经受进一步IHC分析，存储3β-HSD活性染色，并在4℃下洗涤胚胎过夜不会导致在后台的一个显着的提高。 在 3β-HSD活性染色胚胎原位杂交（ISH）分析是可能的。例如，Prox1的转录物的表达是在steroidogen共定位与3β-HSD活性IC组织10。去除了执行3β-HSD活性染色到ISH，胚胎必须固定在4％的PFAT为前和3β-HSD活性测定后，使细胞RNA转录物可以与用于ISH分析期望质量被保留4小时。 通过使用ff1b，CYP11A1，hsdb1，和明星的核糖核酸探针的ISH分析已在interrenal类固醇1,2,11组织的发育研究中得到应用。 ff1b的核糖体探针检测原始interrenal细胞22 HPF，而CYP11A1和明星的检测由24 HPF分化interrenal细胞。相比，3β-HSD组织化学染色仅检测来自28 HPF起分化interrenal组织，ISH分析可用于分析的interrenal特异性基因的频谱中两个原始和分化interrenal细胞。尽管如此，3β-HSD组织化学染色公顷的S是简单，快速和可靠的，用于分析所述分化类固醇interrenal组织的表型的优势，并且因此是用于详细发育研究的强有力的工具，以及大规模的遗传或化学屏幕。根据我们的相关文献复习，不存在能够可靠地通过免疫组化检测在类固醇interrenal组织中的表达特异性抗体。因此，3β-HSD组织化学染色的方法是在划定组织和类固醇合成interrenal组织的细胞形态特别有用。 虽然3β-HSD组织化学染色为分化的类固醇interrenal组织的可视化是有用的，它并不直接报告类固醇合成的能力或interrenal细胞功能。突变或化学影响上游或3β-HSD的下游在类固醇合成级联酶可能不一定与该所述方法被检测到，因为它们可能会或可能不会干扰任一大小或interrenal组织的形态。因此，该协议的用户应牢记，影响某些酶的损伤作用，但interrenal组织形态没有可能不会用这种方法很容易检测。

肾上腺，下丘脑 - 垂体 - 肾上腺轴的重要组成部分，分泌类固醇和类固醇协调平衡和身体应激反应。肾上腺包括外皮层，可分泌类固醇在特定区域的方式，和内髓质，其中合成儿茶酚胺。在硬骨鱼的interrenal腺是在哺乳动物中肾上腺的对应，并且由类固醇合成interrenal和嗜铬细胞，这是分别1-3肾上腺皮质和髓质，功能等价物。已经使用斑马鱼模型进行的研究报告，这两个类固醇和嗜铬细胞谱系是由分子和细胞机制高度类似于那些在哺乳动物1,2-形成。因此，斑马鱼是研究的遗传性疾病，神经内分泌控制和下丘脑 - 垂体 - 肾上腺（interrenal）轴的系统生物学潜在强大的模型。 4,5-转换，和雄烯二酮。 3β-HSD为生物合成的激素类固醇，即孕酮，糖皮质激素，盐皮质激素，雄激素，和雌激素的所有类重要的。两个人3β-HSD同工酶HSD3B1和HSD3B2差异表达6。 HSD3B1在胎盘和外周组织中表达，而HSD3B2在肾上腺皮质和性腺表达。人类HSD3B1和HSD3B2是斑马鱼HSD3B1，这是在interrenal组织和成人性腺表达的共同的直向同源物;斑马鱼hsd3b2是一个母系基因表达的转录的器官前7消失。在整个安装3β-HSD酶活性测定斑马鱼的方案，通过修改利维的方法发达，■通过米兰等描述。上八硬骨鱼类8种冰冻切片。因为组织渗透性和显影斑马鱼的光学透明的，整个安装3β-HSD组织化学可成功地用于固定斑马鱼胚胎和幼虫和具体划定分化interrenal组织。 这个敏感和快速的测定法已应用于各种突变体和morphants展示不同类型interrenal dysmorphogenesis的。所述interrenal3β-HSD活性在其中interrenal组织的规范是通过Ff1b转录因子的特定击倒打乱并作为interrenal分化由Ff1b coregulator Prox1的9,10的击倒影响被降低的胚胎不存在。值得注意的是，3β-HSD活性可在重症早期缺陷的突变体进行检测，如独眼针头和眯 ，其中3β-HSD组织化学界定了interrenal细胞迁移是如何影响11。所述interrenal组织的分化不即使在完全不存在的血液和血管损害。因此，内皮源性信号是如何塑造发展interrenal机关可确定12,13。总体而言，此组织化学测定法已被成功地用于在斑马鱼模型学习规范，分化和类固醇合成的细胞的迁移。因此，它应该是一个高效率和为任何遗传或化学屏幕定位肾上腺和interrenal器官障碍的可靠工具。

<table><tbody><tr><td>Confocal microscope</td><td>Carl Zeiss&amp;nbsp;</td><td>LSM510&amp;nbsp;</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418&amp;nbsp;</td><td></td></tr><tr><td>Glycerol</td><td>USB</td><td>US16374</td><td></td></tr><tr><td>Hyclone Fetal Calf Serum&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>SH30073</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636</td><td></td></tr><tr><td>&amp;beta;-Nicotinamide adenine dinucleotide hydrate</td><td>Sigma</td><td>N1636</td><td></td></tr><tr><td>4-Nitro blue tetrazolium</td><td>Promega</td><td>S380C</td><td></td></tr><tr><td>Nusieve GTG</td><td>Lonza</td><td>50081</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Phenylthiourea</td><td>Sigma&amp;nbsp;</td><td>P7629</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>P4417-100Tab</td><td></td></tr><tr><td>PYREX Spot Plate</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>Reef Salt</td><td>AZOO</td><td>AZ28001</td><td></td></tr><tr><td>trans-Dehydroandrosterone</td><td>Sigma</td><td>D4000</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000M</td><td></td></tr></tbody></table>

visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

要确定如何与pronephric肾小球及新生血管的生成类固醇interrenal组织codevelops中，3β-HSD酶活性测定的双转的Tg（wt1b:GFP）进行LI1;的Tg（kdrl:mCherry）CI5胚胎在34 HPF （ 图1）。在这个阶段，3β-HSD活性阳性类固醇合成组织位于右向中线，并立即尾鳍到pronephric肾小球，而一些类固醇合成细胞开始跨越中线迁移并形成从背视图一个突出边缘。的类固醇合成组织紧密地与背主动脉（DA）和后部主静脉（PCV）相关联。 上的Tg（kdrl:GFP）的3β-HSD酶活性测定胚胎S843允许围interrenal船只的明确界定，包括DA，PCV和interrenal容器（IRV; 图2）。的3β-HSD活性染色的胚胎Vibratome切片进行IHC分析，用于检测所述细胞外基质蛋白纤连蛋白和其下游效应磷酸粘着斑激酶。纤维连接蛋白的围DA沉积是支持IRV增长18是必不可少的。 <img alt="图1" src="/files/ftp_upload/54820/54820fig1.jpg" /> 图1: 整个安装对斑马鱼表达荧光记者进行3β-HSD活性染色。背（左图）和背侧（右图）美景的3β-HSD活性染色的Tg（wt1b:GFP）的LI1; Tg的（kdrl:mCherry）在34 HPF CI5胚胎，它允许可视化interrenal的相对空间分布组织（IR），pronephric肾小球（PG），pronephric管（PT），背主动脉（DA），足背外侧主动脉（LDA）和后部主静脉（PCV）。由于在此阶段interrenal组织的位置，背外侧观点从胚胎的右侧。 D，背; V，腹; A，前壁; P，后路; R，右; L，离开了。比例尺= 50微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54820/54820fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54820/54820fig2.jpg" /> 图2:Vibratome 切片和3β-HSD活性染色 的Tg（kdrl:GFP） 的IHC分析 S843 胚胎 。 34 HPF胚胎进行一个3β-HSD的酶活性测定法，然后vibratome切片。的3β-HSD活性阳性部分是由双IHC染色用兔抗人纤连蛋白（FN; 1/200）和小鼠抗人磷酸粘着斑激酶（pFak; pY397，1/100）。横截面示出了interrenal组织（IR）和其相邻的船只，包括背主动脉（DA），后主静脉（IRV）和后主静脉（PCV）的相对分布。 NT，神经管; NC，脊索; S，体节。比例尺= 50微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54820/54820fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td>股权集中度</td><td>在1000微升</td><td>最终浓度</td></tr><tr><td> A部分: </td><td></td><td></td><td></td></tr><tr><td> DMSO </td><td></td><td> 40微升</td><td></td></tr><tr><td> DHEA </td><td> 10毫克/毫升</td><td> 10微升</td><td> 0.1毫克/毫升</td></tr><tr><td> NAD </td><td> 1.2毫克/毫升</td><td> 10微升</td><td> 12微克/毫升</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> B部分 </td><td></td><td></td><td></td></tr><tr><td> PBST </td><td></td><td> 936微升</td><td></td></tr><tr><td>维生素B3 </td><td> 50毫克/毫升</td><td> 2微升</td><td> 0.1毫克/毫升</td></tr><tr><td> NBT </td><td> 50毫克/毫升</td><td> 2微升</td><td> 0.1毫克/毫升</td></tr></tbody></table> 表1:3β-HSD染色反应的成分。

Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

在斑马鱼的interrenal腺是哺乳动物的肾上腺的硬骨鱼类的对应。此协议引入了如何执行3-β羟基类固醇脱氢酶（Δ5-4异构酶;3β-HSD）的酶活性测定法，其检测在显影斑马鱼分化类固醇合成的细胞。

可视化Interrenal类固醇合成组织及其血管微环境斑马鱼

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

visualizing-interrenal-steroidogenic-tissue-its-vascular

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.9K 次观看.  Tunghai University. 在斑马鱼的interrenal腺是哺乳动物的肾上腺的硬骨鱼类的对应。此协议引入了如何执行3-β羟基类固醇脱氢酶（Δ5-4异构酶;3β-HSD）的酶活性测定法，其检测在显影斑马鱼分化类固醇合成的细胞。 

视频: Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

科研

发育生物学

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

免疫染色磷酸化表位整装斑马鱼胚胎纤毛器官

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

化学

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

幼虫斑马鱼性腺组织的解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

在斑马鱼肿瘤异种移植物中宿主血管反应的Vivo成像和定量

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

癌症研究

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

成年斑马鱼肾脏的解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

生物学

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

通过鱼眼研究糖尿病: 成人 tg (fli: EGFP) 斑马鱼视网膜血管的显微解剖、可视化和分析

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

医学

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

平山准备观察斑马鱼胚胎标本染色，并通过分析全山<em&gt;原位</em&gt;杂交

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...