We thank Matthew O'Connell, Maryam Jahanshahi, and Andreas Jenny for assistance. We thank Istv&#225;n And&#243; for plasmatocyte-specific antibodies, Utpal Banerjee for dome-meso-EBFP2 flies, Julian Martinez-Agosto for antp&gt;GFP flies, and Michael O'Connor for ptth and ptth&gt;grim flies. These methods were developed with support by the Kimmel Foundation, the Leukemia &amp; Lymphoma Society, NIH/NCI R01CA140451, NSF 1257939, DOD/NFRP W81XWH-14-1-0059, and NIH/NCI T32CA078207.

1.循环血细胞浓度<ol><li> 6小时 - 以获得大致相同的发育阶段的幼虫该测定中，通过允许雌性产卵2的一个固定的时间段限制鸡蛋收集。 </li><li>在解剖盘孔填充用1×磷酸盐缓冲盐水（PBS， 表1）收集幼虫。 </li><li>对于每一个幼虫，将10微升1×PBS中在微量离心管在冰上和10μl1×PBS中在一个干净的解剖垫。将解剖盘上的照明体视显微镜基地。 </li><li>通过将它放置在纸巾上干燥个别幼虫将其转移到解剖垫PBS下降之前擦拭。 </li><li>用钳子，轻轻撕开，小心反转角质层释放淋巴。 注:请注意不要划伤或戳的角质层，因为这可能会释放出无柄血细胞29。 </li><li>用移液管，收集来自解剖垫血淋巴。避免同llecting幼虫的碎片，如肥胖的身体。 </li><li>血淋巴添加到微量离心管中，并通过上下抽吸混合。 注意:几个样品可以一次被收集并在冰上保持长达一个小时。 </li><li>可选:将台盼蓝与等份血淋巴样品染色并排除死细胞。算上加台盼蓝，因为它是细胞毒性的3分钟内的细胞。 </li><li>吹打起来，装载10微升到血球室之前向下混合样品。 </li><li>如果使用自动细胞计数，设置细胞大小和圆适当的参数。例如，设定为2μm的最小单元尺寸为22微米的最大细胞大小，和75的圆- 80％的圆度来检测正常，圆形血细胞34。尝试使用不同的参数来检测更大，纺锤状血细胞，如lamellocytes。或者，使用血球手动计数血细胞。 </li></ol> 2.巡回culating血细胞免疫组化<ol><li> 6小时 - 以获得大致相同的发育阶段的幼虫该测定中，通过允许雌性产卵2的一个固定的时间段限制鸡蛋收集。 </li><li>将一个盖玻片在6-或12孔平板的每个孔中。 注:方形盖玻片（22毫米）配合在6孔板和轮盖玻片（18毫米直径）配合在12孔板中。 </li><li>在解剖盘井充满1X PBS收集幼虫。 </li><li>放置5微升1×PBS中在每个盖玻片的中心。 </li><li>将平板上的照明体视显微镜基地。 </li><li>干上组织一个单独的幼虫擦拭，然后将在PBS盖玻片上。 </li><li>用钳子，轻轻撕开，仔细反转角质层。取出幼虫尸体之前，用它来均匀分布的淋巴。 注:请注意不要划伤或戳的角质层，因为这可能会释放出无柄血细胞29。 </li><li> RepeaT代表所有的幼虫，收集每盖玻片1个幼虫血淋巴。允许血细胞附着在盖玻片5 -在室温下室温8分钟，不要超过固定前30分钟。 </li><li>通过加入5微升7.5％的甲醛（ 表1），以每个盖玻片，在室温下15分钟固定的血细胞。 </li><li>洗涤用1×PBS盖玻片三次。提示板和每次冲洗后吸出PBS。为了防止在下一步骤中的透液的流失，使用抽吸器从盖玻片的外周除去过量的PBS。 </li><li>加入100μl透溶液（ 表1），以每个盖玻片在室温20分钟。 </li><li>提示并轻轻拍打板吸出通透的解决方案。 </li><li>稀释使用根据供应商的规范抗体溶液（ 表1）第一抗体。至少添加500μl的初级抗体溶液至盖玻片在12孔板中。在6孔板中添加至少1.5毫升在4℃孵育过夜。 </li><li>除去第一抗体溶液，并用1×PBS在轨道摇床，三次洗涤盖玻片10分钟。 </li><li>根据供应商的规格稀释二级抗体与抗体溶液。至少添加500μl的第二抗体溶液，以12孔板的盖玻片。添加至少1.5毫升6孔板中。在室温下孵育至少2小时。 </li><li>去除二级抗体溶液并用1×PBS在轨道摇床上洗涤盖玻片10分钟，如在步骤2.10，三次。 </li><li>在洗涤步骤中，干净的玻璃显微镜用组织湿巾70％的乙醇（ 见表1）滑动。对于每个盖玻片，置于载玻片上5微升安装缓冲液（ 表1）。 注:两个盖玻片适合在一张幻灯片。 </li><li>吸出最后的PBS洗涤。 </li><li>使用镊子小心从板和地点，INV取出盖玻片erted，在安装缓冲器的顶部。 注:显微镜载玻片可成像之前被储存在4℃。最大存储时间取决于所用的抗体的稳定性。 6-和12孔板可冲洗和再利用。 </li></ol> 3. 在水晶体内细胞黑化<ol><li>以获得大致相同的发育阶段的幼虫该测定中，通过允许雌性产卵的2-6小时的固定时段限制鸡蛋收集。 </li><li>开始之前，设定在60℃的加热源。 注:60℃，10分钟的热循环仪程序（随后是25℃保持）的效果最好，但水浴或其它加热源是足够只要热量被始终如一地均匀地分布在各幼虫。 </li><li>在解剖盘孔填充用1×PBS（ 表1）收集幼虫。 </li><li>一次一个，干上的组织幼虫在PCR管的底部擦拭和地点。将每个幼虫在一个单独的PCR管。 （ 见图3A。） </li><li>对于一致的结果，保证幼虫留在PCR管的底部在4℃，10心寒的管幼虫 - 15分钟和/或轻轻加热前轻敲管。 </li><li>放置在热循环仪（或水浴）的PCR管。热，在60℃下进行10分钟。 </li><li>小心地从PCR管取出幼虫进入解剖盘井充满了新鲜的1X PBS。 </li><li>上的组织干幼虫擦拭并在立体显微镜下安排在成像平面上。 </li><li>一味地由多个个人得分幼虫的图像。 </li></ol> 4.幼虫淋巴腺免疫组化注:淋巴腺距离上背侧大脑略低于一个幼虫的前端大约三分之一的长度。 （参见图3B中的箭头）。淋巴腺侧翼背船只和最容易解剖附着在口呼KS或到大脑。野生型，三龄淋巴腺是非常小的结构;主波瓣大约100 - 200微米的长度。 （参见图4A）。 <ol><li> 6小时 - 以获得大致相同的发育阶段的幼虫该测定中，通过允许雌性产卵2的一个固定的时间段限制鸡蛋收集。 </li><li>淋巴结清扫术<ol><li>对于每个实验条件下，加入1ml 1×PBS中（ 表1）至24孔板的一个孔中。 </li><li> 0.1％的PBST（ 表1）1滴添加到各孔用一次性移液管。 注:洗涤剂，例如吐温20，被添加以降低表面张力，从而允许组织下沉到井的底部。 </li><li>放置板在冰上平。 </li><li>在解剖盘井充满1X PBS收集幼虫。 </li><li>将干净的解剖盘上的照明体视显微镜基地。使用一次性移液管放置SMaL公司升下降0.01％PBST（ 表1）上的垫。转移一个幼虫解剖一个PBST下降。 </li><li>握住幼虫有一对钳约1/4长度从后端，背侧。 </li><li>使用另一双钳抢到角质层前壁立即向持有幼虫镊子。轻轻一拉角质层朝前端，直到口钩暴露。 注意:目的是要剥离的角质层，而不会干扰任何内部结构。 </li><li>松开角质层，同时使用钳剪幼虫两种。取下PBST降后结束。 注:如果角质层没有一路剥下来了口钩，切幼虫在两个取出后结束。用一对镊子的保持角质层的边缘，并使用另一对钳子的通过开口推口钩。这将反转角质层和揭露口钩。这可以被用来作为一种altern吃了解剖方法为好。 </li><li>用一对镊子的牵制对稳定性的角质层（或者腹面角质层，背侧角质层瓣，或两者）。 </li><li>使用另一双钳抓住露出口钩，轻轻地拉出来。 注意:这将角质层从内部结构分开。理想的情况下，眼睛/触角成虫盘，脑，环腺和淋巴腺侧翼背容器将保持附着在口钩。 </li><li>虽然还拿着口钩，小心地取出多余的结构，如唾液腺，脂肪体和肠。 注:如果大脑从口钩分离，淋巴腺可能仍附在并与大脑收集。在这种情况下，保持腹神经索代替口钩。 </li><li>用口钩或腹神经索作为手柄，转移含有淋巴腺在冰上井的解剖复杂，不要超过30分钟固定前。 </li></ol></li><li>固定和免疫组化<ol><li>放置在立体显微镜基的24孔板。 </li><li>使用P200移液器小心地从井里取出PBST。 注:空，邻井暂时存废有用。 </li><li>轻轻加入200微升3.7％的甲醛（ 表1）向下井的侧面，摇动板以确保解剖组织被完全淹没。 </li><li>返回板冰上30分钟。如果淋巴腺表达荧光蛋白（S），保持覆盖以防止光漂白的板。 </li><li>使用P200移液器小心取出固定液。 </li><li>通过加入200μl的1×PBS中的井和放置板在定轨摇床上5分钟，在室温下洗涤。使用P200移液器小心取出PBS。 注:固定淋巴腺可以在冰留在PBS直到淋巴腺所有实验条件是dissected并固定。 </li><li>重复洗涤步骤两次以上。 </li><li>加入200μl通透溶液（ 表1）到孔中。上在室温45分钟轨道摇床设定的板。 注意:45分钟是"黄金标准"淋巴腺通透但作者都只有20分钟后成功。 </li><li>删除与P200移液器的解决方案。 </li><li>稀释使用根据供应商的规范抗体溶液（ 表1）第一抗体。添加300微升初级抗体溶液至孔，并确保解剖组织被完全淹没。在4℃孵育过夜。 </li><li>使用P200移液器除去初级抗体。 </li><li>通过加入200μl的1×PBS中的井和放置板在定轨摇床上10分钟，在室温下洗涤。使用P200移液器小心取出PBS。 </li><li>重复洗涤步骤两次以上。 </li><li>二次稀释一根据供应商的规格与抗体溶液ntibody。加入300微升二抗溶液至孔，并确保解剖组织被完全淹没。孵育在轨道摇床上于室温至少2小时。 </li><li>使用P200移液器去除二级抗体并在步骤4.3.12＆4.3.13描述洗净。最后一次洗涤后不要取出PBS。 </li></ol></li><li>淋巴腺安装<ol><li>清洁的玻璃显微镜载玻片用70％的乙醇（ 见表1）和组织湿巾。 </li><li>对于每种实验条件下，放置于载玻片上安装缓冲液（ 表1）中的一个的下降。 注:两个条件适合在一张幻灯片。安装缓冲体积取决于待安装淋巴腺的数目。使用2微升约18 - 20淋巴腺，1μl的10 - 12日，和0.5微升5或更小。 </li><li>广场上的照明立体底座的显微镜载玻片。 </li><li>用于一次最多两个条件下，从井传输所有淋巴腺到安装缓冲用钳子，使用口钩或腹神经索作为手柄。 </li><li>空间中的解剖组织均匀的圆形或矩形形状，扩展的过程中安装缓冲器。 </li><li>对于每个解剖组织，独立，背船只下滑动钳一个夹钳和朝向安装缓冲外围轻轻一拉。 注意:这将引起淋巴从解剖组织的其余部分进行压盖和扁平玻璃上的淋巴结。 </li><li>使用镊子的一个夹钳和锯切运动，切淋巴腺和大脑之间的背血管。 </li><li>移动至解剖组织的其余部分淋巴腺的相对侧，在缓冲的最外边缘。 注:不需要解剖组织最终将形成围绕淋巴腺周边并作为对whic支持小时盖玻片将休息。 </li><li>重复，直到所有的淋巴腺从解剖组织分离，保留不需要的剖分组织在中心放置的一个。 </li><li>以两个手指之间的盖玻片。检查盖玻片是免费的灰尘和指纹。如有必要，使用纸巾擦拭和70％的乙醇清洁盖玻片。 </li><li>确保盖玻片的边缘平行于玻璃载片的边缘，小心地放在安装缓冲器盖玻片。 注:显微镜载玻片可成像之前被储存在4℃。最大存储时间取决于所用的抗体的稳定性。 24孔板可冲洗和再利用。 </li></ol></li></ol> 5.成像<ol><li>固定图像和血细胞根据制造商的操作手册在20X或更高的放大倍率装在一个适当的标准荧光或共聚焦显微镜淋巴腺。在支架上的图像整体的幼虫根据制造商的操作手册以2倍的放大倍率ARD体视显微镜。 </li><li>按照软件供应商的说明去卷积，如果需要的话。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>解</td><td>组成</td><td>存储</td><td>注释</td></tr><tr><td rowspan="5"> 1X PBS </td><td> 200毫克/升的氯化钾</td><td rowspan="5">室内温度</td><td rowspan="5"></td></tr><tr><td> 200毫克/升磷酸二氢钾</td></tr><tr><td> 8000毫克/升氯化钠</td></tr><tr><td> 1150毫克/升的磷酸氢二钠</td></tr><tr><td>卫生署2 O </td></tr><tr><td>固色剂</td><td> 3.7％或7.5％的甲醛在1X PBS </td><td>室温在黑暗中</td><td>甲醛是有毒的。 </td></tr><tr><td rowspan="3">通透的解决方案/抗体稀释剂</td><td> 0.4％的Triton </td><td rowspan="3"> 4℃ </td><td rowspan="3">标准公式使用0.4％的Triton但作者使用0.1％吐温20以成功。使用根据供应商的推荐浓度稀释小学和中学的抗体。 </td></tr><tr><td> 5％牛血清白蛋白，正常山羊血清，或正常驴血清</td></tr><tr><td> 1X PBS </td></tr><tr><td> 70％的乙醇</td><td>蒸馏水中的70％的200度乙醇</td><td>室内温度</td><td></td></tr><tr><td rowspan="4">安装缓冲</td><td> 0.5％N-丙基没食子酸</td><td rowspan="4"> 4℃在黑暗中</td><td rowspan="4">棓酸正丙酯是有害的。 DAPI是一种诱变剂。 </td></tr><tr><td> 80％甘油</td></tr><tr><td>可选:1μg/ ml的DAPI（4&#39;，6-二脒基-2-苯基） </td></tr><tr><td> 1X PBS </td></tr><tr><td> 0.1％PBST </td><td> 0.1％吐温20在1×PBS中</td><td>室内温度</TD&gt; <td></td></tr><tr><td> 0.01％PBST </td><td> 1:10稀释0.1％的PBST </td><td>室内温度</td><td></td></tr></tbody></table> 表1.本议定书中所使用的解决方案。

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作者宣称，他们没有竞争或经济利益。

在遗传或环境的改变，这里所描述的四种方法可单独或联合用于分析诸如信令，存活，增殖和分化的造血过程中不同的过程果蝇造血是一个动态过程。每只动物血细胞的数量增加35和发育过程中的淋巴腺的结构和基因表达的变化32。之前执行这些测定法，因此，它是通过使雌性产卵的固定时间量，并确认所期望的幼虫的发育阶段来限制蛋集合的关键。对于更短的蛋集合（小于6小时）或对于其中女性是不健康的或稀少的情况下，产蛋可以通过在食品创建缝隙得到鼓励。这可通过用乙醇清洗刮刀除去食品的一部分来实现。如果多余的液体食品的积累，利用组织湿巾沐浴液体。 （参见图1A）。 单独，这里所描述的方法有局限性。例如，测量血细胞循环浓度捕获血细胞存活或增殖变化，但没有提供关于血细胞谱系分布或可能因之遗传或环境改变任何血统混乱的信息。相反，循环血细胞的免疫组化显示在特定的血淋巴血细胞谱系，但只有在相对的，不是绝对的，数字的变化。 在体内的晶元黑色素难以定量水晶细胞分布是可变的，无柄血细胞是动态的8,10。相反，多个人应得分晶胞黑色素盲目。最后，观察在一个血细胞车厢所作不一定持有其他车厢如此。 当一起使用时，这些测定法可以适用于区分基因抽动改变调节增殖或存活从那些调节基因表达或分化。例如，一个遗传改变可以增加血细胞的增殖，使得血细胞浓度的增加，但血细胞谱系的相对比例保持不变。另外，遗传改变能促进整体血细胞浓度没有影响血细胞中分化变为具体的谱系。晶体细胞群可以快速，轻松地询问使用体内黑色素的方法，促进遗传筛选和遗传相互作用的研究。这种方法可以在佐证可以使用与利用酚氧化酶原 1（PPO1，也称为黑细胞，BC）的突变等位基因的基因研究或利用晶体细胞特异性抗体免疫组织化学测定。幼虫淋巴腺可以用来解决有关造血细胞过程类似的问题。众多信号通路涉及在建立和维持淋巴腺及其主要区域。每个区域都有幼虫造血功能不同的基因表达和功能。此外，利用淋巴腺可以揭示淋巴结区域或淋巴结内血细胞自主和非自主功能。 为研究果蝇造血相关方法已经在全面的基于文本的资源，编译最近才26,27。虽然不可缺少的领域，这些资源是在范围的限制。用于测量血细胞的浓度的方法，例如，不包括在内。写方法，尤其是那些描述解剖技术，可以挑战迅速掌握。额外的贡献已经作出，提供视觉资源来协助方法，但在数量和范围28,29仍然被限制。此处所描述的方法，同时，也并不全面，添加到资源阿瓦伊拉布勒在果蝇造血功能的研究提供帮助。 我们提供的修改和替代现有的协议。例如，有几个优点测量血细胞浓度用自动细胞计数器，而不是一个人工血球，包括提高的速度，缓和，和客观性。根据我们的经验，使用水浴加热为液晶单元的可视化的幼虫导致广泛不同的结果，特别是如果幼虫在它们所提出的小瓶加热。在个别的PCR管加热幼虫产生较少变量和更可重复的结果。这里所描述的淋巴结清扫方法，虽然先前在一份书面协议概述26，为现有的目视参考28的替代品。最后，如果获得了共焦显微镜是有限的，我们建议标准荧光图像的去卷积可能是共焦图象的合适替代品。 Deconvoluti于算法删除或重新分配失焦光通过常规荧光显微镜拍摄的，提高的分辨率和在原理对比类似于消除失焦光通过共聚焦显微镜的，并且可以适用于2维和3-维（Z堆叠）图像。对于一些应用，如这里证明的，去卷积可以显着提高与常规荧光显微镜拍摄的图像。

调节转录因子和信号转导通路即坐标造血系统的在血液系统疾病的发展，而且故障复杂机制仍然知之甚少。这些转录因子和信号通路，以及它们的调节，是高度果蝇和哺乳动物造血1-5之间是保守的。因此， 果蝇造血系统代表一个优秀的遗传模型来定义的分子机制控制造血和潜在的血液系统疾病。 哺乳动物类似， 果蝇产生在空间和时间上不同的造血的相位血细胞，称为血细胞。传统上， 果蝇造血被认为在胚胎中胚层和在幼虫淋巴腺被限制在相。最近的研究提供的证据表明，血细胞生成，也会发生在幼虫无梗CLU讲演者，并在成人腹部6-8。所有造血阶段产生两种类型的血细胞成熟的:浆细胞和晶体细胞。浆细胞是参与吞噬作用，先天免疫，和伤口愈合的巨噬细胞样细胞。水晶细胞含有黑色素，在昆虫免疫反应和伤口愈合使用的反应所需的亲phenoloxidases。幼虫造血可以产生第三成熟血细胞类型，称为lamellocyte，响应于如寄生蜂感染9,10-某些免疫挑战。 Lamellocytes是功能，与浆细胞和水晶结合的细胞，封装和消除在果蝇幼虫黄蜂奠定鸡蛋大，贴壁细胞。在不存在寄生的，lamellocytes未在野生型幼虫找到。黑色素群众像白化，封装蜂卵;许多果蝇突变株培养在没有寄生的黑色素群众。 lamello的存在核细胞和/或黑色素群众可以指示造血异常。事实上，黑色素质量表型已经被用于鉴定参与造血11-14基因和途径。 幼体造血系统是最广泛研究日期。它是由血细胞在血淋巴中循环的，角质层下图案化无梗血细胞群集，和血细胞驻留在淋巴腺。淋巴腺是一系列附连到背脉管双边瓣。淋巴腺的每个主叶被分成三个主要区域。最外面的区被称为皮层区和包含成熟的血细胞。最里面的区域被称为髓区并且包括静态血细胞前体。第三区，后信令中心，是在淋巴腺充当一个干细胞样的龛的基部一小群细胞。早期的工作建立关键功能的缺口15-18 </suP&gt;，刺猬19,20，JAK-STAT 18，和无翅21的活动来调节幼虫淋巴腺发展。最近的研究已经表明，BMP 22，FGF-RAS 23和河马24,25信令也幼虫淋巴腺内起作用。 这里概述四幼虫造血测定法描述1）测量循环血细胞浓度，定义为每单位体积的细胞数，2）分离和定影循环血细胞对于免疫组化，3）可视晶体细胞在体内 ，和4）解剖，定影，和安装淋巴腺进行免疫组化。这些测定可用于造血读数来评估的功能和在幼虫造血系统的信号通路的规定。虽然这些方法已经在该领域以前使用的，这些试验的视觉文件已经开始最近才8,26-30。这里列举了几个出版物帮助FUL资源描述类似的方法和造血标记26,31-33。此外，控制和海盗是淋巴腺基底膜的有效指标。

<table border="0" cellpadding="0" cellspacing="0" width="804">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">PBS tablets</td>
			<td style="width:176px;">MP Biomedicals</td>
			<td style="width:163px;">2810305</td>
			<td style="width:236px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">dissecting dish</td>
			<td style="width:176px;">Corning</td>
			<td style="width:163px;">7220-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">microcentrifuge tube</td>
			<td style="width:176px;">Denville</td>
			<td style="width:163px;">C2170</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:229px;">silicone dissecting pad, made from Sylgard 184 kit</td>
			<td style="width:176px;">Krayden (distributed through Fisher)</td>
			<td style="width:163px;">NC9644388 (Fisher catalog number)</td>
			<td style="width:236px;">Made in petri dish by mixing components of Sylgard elastomer kit according to manufacturer instructions.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">stereomicroscope</td>
			<td style="width:176px;">Morrell Instruments (Nikon distributor)</td>
			<td>mna42000, mma36300</td>
			<td>Nikon models SMZ1000 and SMZ645</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">tissue wipe</td>
			<td style="width:176px;">VWR</td>
			<td style="width:163px;">82003-820</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">forceps</td>
			<td style="width:176px;">Electron Microscopy Sciences</td>
			<td style="width:163px;">72700-DZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">p200 pipette</td>
			<td style="width:176px;">Eppendorf</td>
			<td>3120000054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Countess Automated Cell Counter</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:163px;">C10227</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Countess cell counting chamber slides</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:163px;">C10283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">hemocytometer</td>
			<td style="width:176px;">Hausser Scientific</td>
			<td style="width:163px;">3200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">trypan blue stain</td>
			<td style="width:176px;">Life Technologies</td>
			<td style="width:163px;">T10282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">formaldehyde</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">BP531-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Triton</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">BP151-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Tween 20</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">BP337-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">bovine serum albumin</td>
			<td style="width:176px;">Rocky Mountain Biologicals</td>
			<td style="width:163px;">BSA-BSH-01K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">normal goat serum</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:163px;">G9023-10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">normal donkey serum</td>
			<td style="width:176px;">Sigma</td>
			<td style="width:163px;">D9663-10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">200 proof ethanol</td>
			<td style="width:176px;">VWR</td>
			<td style="width:163px;">V1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">N-propyl gallate</td>
			<td style="width:176px;">MP Biomedicals</td>
			<td style="width:163px;">102747</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">glycerol</td>
			<td style="width:176px;">VWR</td>
			<td style="width:163px;">EM-4750</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">DAPI (4&#8217;,6-diamidino-2-phenylindole)</td>
			<td style="width:176px;">Fisher</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">6-well plate</td>
			<td style="width:176px;">Corning</td>
			<td style="width:163px;">351146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">12-well plate</td>
			<td style="width:176px;">Corning</td>
			<td style="width:163px;">351143</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">microscope cover glass, 22 mm square</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">12-544-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">microscope cover glass, 18mm circular</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">12-545-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">glass microscope slides</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">22-034-980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">thermal cycler</td>
			<td style="width:176px;">Eppendorf</td>
			<td>E950010037</td>
			<td>Mastercycler EP Gradient S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">PCR tubes</td>
			<td style="width:176px;">USA Scientific</td>
			<td style="width:163px;">1402-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">24-well plate</td>
			<td style="width:176px;">Corning</td>
			<td style="width:163px;">351147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">disposable transfer pipet</td>
			<td style="width:176px;">Fisher</td>
			<td style="width:163px;">13-711-9AM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">fluorescence microscope</td>
			<td style="width:176px;">Zeiss</td>
			<td style="width:163px;"></td>
			<td>Axio Imager.Z1</td>
		</tr>
	</tbody>
</table>

methods to examine the lymph gland and hemocytes in drosophila larvae

果蝇 果蝇和哺乳动物造血系统之间有许多共同点存在，即使果蝇缺乏淋巴系表征哺乳动物适应性免疫，和哺乳动物造血发生在时间和空间上不同的阶段产生多种血细胞谱系。这两个系统都保持血液祖细胞与扩大或更换成熟谱系的水库。造血系统允许果蝇和哺乳动物以应对和适应免疫的挑战。重要的是，用于控制生成，维护，和造血系统的功能的转录调节和信号传导途径是从果蝇保守到哺乳动物。这些相似性允许果蝇被用于遗传模型造血发育和疾病。 下面我们详细分析研究果蝇幼虫的造血系统。在特别是，我们概括的方法来测量血细胞数量和浓度，体内可视化的特定成熟细胞系，并在循环和造血器官进行对血细胞免疫组化。这些分析可以揭示基因表达的变化的细胞过程，包括信令，存活，增殖和分化，并且可以用于研究各种关于造血的问题。在果蝇中可用的遗传工具相结合，这些测定法可以用于评估在所定义的遗传改变的造血系统。尽管没有特别这里概述，这些试验也可以用来检查环境的改变，如感染或饮食的影响，对造血系统。

循环血细胞浓度血细胞数在整个幼虫发育35增加。为了说明，该方法检测血细胞数量和浓度的差异，无论生物原因，我们测量延迟和非延迟的幼虫血细胞的浓度。促前胸腺激素（PTTH）由PTTH产生神经元（PTTH&gt;严峻）的遗传消融的损失产生于幼虫发育36的延迟。对于每个基因型，对于在25℃升高至少8个人幼虫在方案1中所述，测定血细胞的浓度。在2小时的鸡蛋收集后120小时，每延期幼虫的平均血细胞浓度（PTTH&gt;严峻）小于每个控制幼虫（PTTH）的平均浓度为血细胞。只有9天之后确实每控件延迟幼虫的方法，平均血细胞浓度（ <st荣&gt;图1B）。其中使用该方法来检测血细胞的浓度的差异的另一个例子已发表37。 从自动细胞计数器拍摄的图像显示一个干净的，可取的血淋巴样品和含有碎片，这可能会导致不准确的测量（ 图1C）的样品。最小和最大的小区尺寸分别设为2微米和22微米。圆被设定为75-80％的圆度。这些参数的目的是作为指导原则，应凭经验优化。 循环血细胞免疫组化表达绿色荧光蛋白（GFP）循环血细胞收集，固定，并孵育特定plasmatocyte抗体（P1A和P1B，伊什特万ANDO）31的混合物作为在协议2中记载（ 图2）。图像拍摄于一个标准的荧光显微镜显示不变，并约束迭代反褶积之后。在这种情况下，解卷积没有大幅度提高图像质量。 在水晶体内细胞黑化幼虫如在协议3（ 图3A）描述放置在PCR管之前热诱导液晶单元黑色素的底部。红色箭头指示管，其中幼虫从底部太远，可能被不均匀地加热。横跨各个幼虫的热分布不均匀的晶体细胞的幼虫内的黑色素增加变化。 成像在一个标准的体视显微镜野生型幼虫表示无柄簇白化结晶细胞热暴露后的典型模式（图3B）。在淋巴腺变暗的晶体细胞有时可见。 幼虫淋巴腺免疫组化第三龄幼虫淋巴腺进行解剖，固定，并安装成在协议4描述在标准荧光显微镜拍摄的微分干涉对比（DIC）图像示出了初级和次级淋巴腺瓣侧翼背容器（ 图4A）。 其中延髓区和后信号被中心遗传标记具有增强蓝色荧光蛋白（EBFP2）和GFP一个淋巴结，分别与针对Notch胞内结构域抗体（C17.9C6，发育研究杂交瘤细胞银行）的染色在协议描述被采取了一个标准的荧光显微镜4. Z堆栈图像。单台最大INT<html>密度投影图像出现没有改变，约束迭代卷积（ 图4B）之后。在这种情况下，解卷积显着提高图像的质量和细节。 <img alt="图1" src="/files/ftp_upload/54544/54544fig1.jpg" /> 图1.循环血细胞浓度。A）标准的果蝇中的一部分被删除（右）在2小时的鸡蛋收集期。B）每发育迟缓幼虫的平均血细胞浓度（PTTH&gt;严峻）是低于促进产蛋每次产卵（AEL）后的120小时防治幼虫（PTTH）的平均浓度为血细胞。每延期幼虫的平均浓度为血细胞接近控制水平9天AEL。误差棒代表±SEM C）的血淋巴样品无（左）和具有（右）的碎屑。刻度条表示为0.1mm。HREF ="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54544/54544fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54544/54544fig2.jpg" /> 图2.循环血细胞免疫组织化学。血细胞基因表达GFP收集并固定在盖玻片。具体plasmatocyte抗体被用于染色浆细胞（红色）。 DAPI染色示于蓝色。未改变的图像拍摄用荧光显微镜（左）。解卷积（右）后的相同的图像。标尺代表50微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54544/54544fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54544/54544fig3.jpg" /> 图3. 体内 </em&gt;水晶细胞黑化。A）幼虫进行单独的PCR管加热前放置。红色箭头指示幼虫不是在管的底部B）中加热后的一个典型的液晶单元黑化模式，这有时揭示淋巴腺（箭头;所示背侧，前壁是左侧）。比例尺为1毫米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54544/54544fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54544/54544fig4.jpg" /> 图4.幼虫淋巴腺免疫组织化学。A） 中示出的背脉管（DV），主波瓣（1°），和副瓣（2°）的第三龄幼虫淋巴腺的DIC图像。比例尺代表100微米。B）代表第三龄幼虫淋巴腺˚FROM幼虫基因表达在后信号中心延髓区和GFP EBFP2。淋巴腺用针对Notch胞内结构域（红色）的抗体染色。未改变的图像拍摄用荧光显微镜（顶部）。后去卷积（底部）相同的图像。比例尺代表20微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54544/54544fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae at JoVE.com

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

果蝇和哺乳动物造血系统有许多共同的特点，使果蝇有吸引力的遗传模型来研究造血功能。这里，我们证明了免疫组化的主要造血幼虫器官解剖和安装。我们还描述的方法来测定各种幼虫造血车厢，包括循环血细胞和无柄水晶细胞。

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

methods-to-examine-the-lymph-gland-and-hemocytes-in-drosophila-larvae

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

12.7K 次观看.  The Icahn School of Medicine at Mount Sinai.  果蝇和哺乳动物造血系统有许多共同的特点，使果蝇有吸引力的遗传模型来研究造血功能。这里，我们证明了免疫组化的主要造血幼虫器官解剖和安装。我们还描述的方法来测定各种幼虫造血车厢，包括循环血细胞和无柄水晶细胞。 

视频: Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Stentor再生的研究方法

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

解冻、培养和冷冻保存的嗜酸性粒细胞系

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

检测果蝇黑色素的内分泌干扰的方法

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

斑马鱼胚胎和幼虫的整个山免疫性化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...