我们要感谢马克·伦纳德·卡尔顿身临其境的媒体工作室视频制作和编辑博士和马丁​​·贝特朗·卡尔顿身临其境的媒体工作室/神经再生实验室的可视化模型援助的专家技术援助。作者非常感谢玛丽莲Keaney博士和她的专职工作人员，在大学渥太华动物保护和兽医服务的专业意见。这项工作是由加拿大卫生研究院（CIHR研究所，澳门币62826）SALB，CIHR老龄研究所和战略的培训计划，在卫生研究/ CIHR培训的神经退行性脂类组学计划（TGF 96121）SALB和SF，加拿大基金会创新，安大略省创新信托SF，SF和Autodesk研究以SF。 ACM收到CIHR Banting和Best博士奖。 NV获得一个专业奖学金后，从人口老龄化和的CIHR培训计划在神经退行性脂类组学研究所。

1。准备试剂<ol><li>对于无菌阴道灌洗，高压灭菌双蒸水（DDH 2 O），并存储在一个密封的容器中，室温，直到需要。 </li><li>对于细胞学评估，结晶紫粉0.1克至100毫升DDH 2 O拌匀。结晶紫染色（0.1％）可被存储在一个密封的容器中，在室温下，直到需要。 </li></ol> 2。收集阴道细胞（阴道灌洗） <ol><li>胶乳灯泡放置在无菌的200μl的尖端的端部和绘制最多约100微升无菌DDH 2 O的前端中使用的灰度作为准则的体积。 </li><li>鼠标抬起她的笼子笼斗（盖），并把她与她的后腿/后端向您。 </li><li>紧紧抓住尾巴和提升的后端。鼠标现在只有她的前爪抓料斗。在这一点上，鼠标可能会排尿。如果是这样的，等到排尿站。如果有尿留在阴道的入口，你可能想使用一个单独的尖端（ 即 ，不是你的样品采集头）冲洗开幕多余的DDH 2 O。 </li><li>将在开幕式的阴道，注意不要穿透孔，阴道和宫颈的刺激能诱发假孕大鼠1,2的DDH 2 O填充尖。最近的报告表明，老鼠是不容易受到这种影响仍然应注意的侵袭程度，以尽量减少重复分析3。 </li><li>轻轻地踏下灯泡，驱逐一季度的一半量的水（约25-50微升）在开幕阴道管。无尖插入液体会自发地吸进运河。缓慢地释放在灯泡上的压力。流体将撤回到尖。避免释放压力的速度太快，以防止误吸液到灯泡。过滤的尖头可能是有用的用于此目的。 </li><li>重复前面的步骤使用相同的尖端，灯泡，和流体的4-5倍，在一个单一的样品，以获得足够数量的细胞。 </li><li>流体放置在玻璃片上，并允许涂抹标本在室温下完全干燥。发情涂片干燥后，可立即染色或存储，并在以后的日子染色。 </li></ol> 3。细胞学染色结晶紫* 4 <ol><li>将在科普林氏jar文件（或其他可比较的染色容器）中含有的结晶紫染色1分钟干滑动。 </li><li>删除科普林氏的第二个罐子DDH 2 O洗净幻灯片DDH 2 O为1分钟。重复以上步骤。 </li><li>删除多余的DDH 2 O幻灯片中一个轻型的组织，雨刮器的边缘，避免接触涂片染色。 </li><li>移液器约15微升的顶部的涂片和盖的甘油打滑。或者，其他组织学安装试剂可以利用，以获得一个更永久的，非扩散染色。 </li></ol> *这里描述的染色方法是最简单的程序，可以在任何实验室进行。其他的方法可以提供更多的细节。例如，采用巴氏染色法，有核上皮细胞的成熟度来区分不太成熟的细胞染色的粉红色或橙染色的绿松石和更成熟的细胞。这些差异可以被用来举办提前或延迟发情前期。 4。阴道细胞学检查<ol><li>检查涂片，光镜下观察，以确定细胞类型。结晶紫从细胞中扩散，随着时间的推移使用甘油时，盖片染色后镜检应立即做。在分析时，应采取显微照片记录细胞学检查。 </li><li>通过检查的entir开始Ë涂抹在较低的放大倍率。选择有代表性的区域并移动到一个更高的放大倍率。您将看到角化鳞状上皮细胞，白细胞，和/或有核上皮细胞（ 具有代表性的结果 ， 图1A-C）。细胞存在的比例将允许你确定你的鼠标动情期，在取样时（ 具有代表性的结果 ， 图1D-G）和她的激素水平（ 讨论 ， 图2）。 </li></ol> 5。代表性的成果 细胞学检查：三种主要的类型的细胞中可检测到阴道涂片样品：（1）有核上皮细胞（ 图1A），（2）角化鳞状上皮细胞（ 图1B），和（3）白细胞（ 图1C）。有核上皮细胞胞浆淡染，较暗的彩色质膜，以及一个椭圆形的核（ <strONG&gt;图1A）。角化鳞状上皮细胞是均匀地染色，更多边形的形状比他们的有核上皮前辈，和没有细胞核（ 图1B）。多形核白细胞，上皮细胞可区别于其形状不规则，深染多态性核，和小尺寸（ 图1C中 ，黑色箭头所示）。如果尿污染本在涂片，尿酸结晶很容易检测到由异种的任何预期类型的细胞（ 图3）的结晶结构体。如果发生这种情况，并不起眼的检测主要的细胞类型，涂抹应该被丢弃，而不是用于举办目的。 分段：涂片中观察到的细胞类型的相对比率上的一天的样品收集（ 图1D-G），可以用来识别你的鼠标的动情周期的阶段。在发情前期，细胞几乎完全集群的圆形，形成有核上皮细胞（ 图1D，代表细胞由白色箭头指示）。在发情期，细胞主要是角化鳞状上皮细胞，存在在密密麻麻的集群（ 图1E，代表细胞的箭头指示）。在发情后期，小深染白细胞占主导地位（ 图1F，代表电池用黑色箭头表示）。角化鳞状上皮细胞中可以观察到，往往在片段，（ 图1F，代表性的细胞用黑箭头表示）。在动情，罕见角化鳞状上皮细胞仍可能存在（ 图1G，代表性的细胞用黑色箭头表示），白细胞仍然占主导地位（ 图1G，代表性的细胞用黑色箭头表示）。发情后期可区别于发情间期核上皮细胞的外观在发情间期（ <stron<html> G&gt;图1G，代表细胞的白色箭头指示）。 <img src="/files/ftp_upload/4389/4389fig1.jpg" alt="图1" /> 图1。阴道涂片的细胞学评估可以用于识别动情期，三个主要类型的细胞中检测到阴道涂片样品的：（A）的有核上皮细胞，（B）的角化鳞状上皮细胞，和（C）的白细胞。涂片这些类型的细胞中存在的比率，可以使用（D）的动情前期，（E）的发情期，（F）的发情后期，或（G）的发情间期中描述的有代表性的结果 ，以确定小鼠。黑色箭头代表角化鳞状上皮细胞E，F和G点。黑色箭头C，F和G点代表leykocytes。在D和G的亮点代表的白色箭头核上皮细胞。 文件，“ALT =”图2“/&gt; 图2。阴道涂片细胞学检查反映潜在的内分泌活动。还提供了详细讨论。 <a href="http://www.jove.com/files/ftp_upload/4389/4389fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img src="/files/ftp_upload/4389/4389fig3.jpg" alt="图3" /> 图3。尿酸结晶可能会出现下面的结晶紫染色尿污染的样本 （A）的晶体是透明的，可以是各种尺寸（箭头和盒装（B）中放大的区域）。没有在这一领域存在的细胞。如果字段内的污染进行细胞学染色尿酸结晶存在，它可能难以准确地确定细胞类型，涂抹应该被丢弃。比例尺= 50微米。

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrous+cycle+modulates+ovarian+carcinoma+growth.">Estrous cycle modulates ovarian carcinoma growth.</a> Clinical cancer research : an official journal of the American Association for Cancer Research. 15, 2971-2978 (2009).</li><li>Jemiolo, B., Harvey, S., Novotny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+the+Whitten+effect+in+female+mice+by+synthetic+analogs+of+male+urinary+constituents.">Promotion of the Whitten effect in female mice by synthetic analogs of male urinary constituents.</a> Proceedings of the National Academy of Sciences of the United States of America. 83, 4576-4579 (1986).</li></ol>

作者宣称没有利益冲突。所有的动物实验均严格按照渥太华大学动物保健委员会和加拿大动物保护协会的准则和规定。

细胞类型学的这些变化是潜在的内分泌活动的指标。动情前期阶段的动情周期对应的月经周期的5人卵泡阶段定义由排卵前增加循环中17-β-雌二醇的水平6，以及一个小的浪涌在催乳素7（ 图2，发情前期，左图）。的增加在17-β-雌二醇的间接刺激促性腺激素释放激素神经元的，反过来，激活响应在垂体前叶细胞释放促黄体激素和促卵泡激素的循环8,9（ 图2的下丘脑和隔垫，发情前期，左图）。取自动物在发情前期阴道分泌物涂片检查，细胞几乎完全是椭圆形的，有核上皮细胞（ 图1D，图2中 ，动情前期，右图）。高峰，卵泡刺激HORM1电平信号排卵进入发情期10,11。在发情期间，17-β-雌二醇水平下降和催乳素水平峰值6,7（ 图2，发情期，左侧面板）。阴道脱落细胞涂片的特点是几乎是唯一的检测不规则形角化鳞状上皮细胞通常在团块（ 图2，图1E，发情期，右图）。进入发情后期恰逢连续上涨的孕激素水平6，对应于开始的12人黄体期（ 图2，动情后期，左侧面板）。由于孕激素水平开始上升，在17-β-雌二醇水平在黄体激活6,13,14（ 图2，动情后期，左侧面板），有一个小高潮。在这个阶段，阴道分泌物涂片检查中存在的细胞类型是零散的，角化上皮细胞和较深的染色白细胞（FIGURË1F， 图2，动情后期，右图）。最后，发生进入发情间期的小鼠和循环黄体酮水平峰值6，对应于人类晚黄体期12。回归的黄体导致随后急剧下降，孕激素水平15,16（ 图2，间情期，左侧面板）。白细胞中占主导地位涂片在动情。角化上皮细胞的频率降低和成核的上皮细胞开始被检测到之前，过渡到动情前期（ 图1G，图2，求偶期，右面板）。 总之，这个简单的，日常的协议可用于生育状况没有改变，如果采取以下的预防措施，估计每天荷尔蒙的波动，并建立实验小鼠的动情期。抽样应在不超过每日一次使用的非侵入性的协议递减ribed相比，阴道，愿望和搅拌，反复渗透。这可能会导致阴道刺激导致的炎症反应17白细胞和其它类型的细胞中产生的，是本涂片可能混淆细胞学评估。此外，即使在殖民地安置女性，这是正常的扩展发情间期和发情阶段，在不同的鼠标，以及诱导休情期18和识别的解释是非常有用的激素影响生殖，性别，疾病的研究。随着年龄的增长和住房群内差异（个人或集团-房屋）的女性7,19-21还引入了变异周期长。女性女性，只有殖民地坐落在停止循环，并进入虽然循环可以重新设置曝光网箱预处理引起男性尿液循环24,25长期动情18,22,23的状态。因此，要建立个别升周期长度的给定的鼠标，建议每日执行这里描述的非侵入性的评估，小心使用，直到观察到两个完整的周期。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录＃ </td></tr><tr><td>无菌200微升吸头</td><td> Diamed </td><td> E340901 </td></tr><tr><td>乳胶灯泡（1毫升） </td><td>费舍尔</td><td> 03-488-21 </td></tr><tr><td>显微镜幻灯片</td><td>费舍尔</td><td> 12-550-15 </td></tr><tr><td>结晶紫染色（25克） </td><td>费舍尔</td><td> C581-25 </td></tr><tr><td>轻型组织雨刷的</td><td> VWR </td><td> 82003-820 </td></tr><tr><td>甘油</td><td>费舍尔</td><td> BP229-1 </td></tr><tr><td>显微镜盖玻片（22x30） </td><td>费舍尔</td><td> 12-544A </td></tr></tbody></table>

performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification

在啮齿类动物的生殖状态评估是一种快速的方法不仅可用于生殖功能障碍的研究，但也需要生产新的小鼠模型的疾病和调查的激素调节组织变性（或再生）病理挑战。小鼠生殖或发情周期分为4个阶段：发情前期，发情期，发情后期和动情。定义卵巢激素17-β-雌二醇和孕酮的循环水平的波动，促性腺激素促黄体生成和促卵泡激素，黄体生成激素催乳激素信号通过这些生殖阶段过渡。小鼠阴道内的细胞类型学的变化反映了这些潜在的内分泌活动。每日评估有核上皮细胞，角化鳞状上皮细胞，和白细胞阴道涂片中存在的相对比例可以用于识别小鼠发情阶段。程度的侵袭，但是，受聘于收集这些样本可以改变生育状况和引起的炎症反应，可以混淆涂片细胞学检查评估。在这里，我们描述了一个简单的，非侵入性的协议，可以用来确定一个雌性小鼠的动情周期的阶段没有改变她的生殖周期。我们详细介绍了如何区分通过收集和分析阴道分泌物涂片检查的主要细胞类型学在发情周期的四个阶段，我们可以解释这些变化是如何与内分泌状态。

Watch this Scientific Journal Video about Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification at JoVE.com

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

在这里，我们介绍了如何识别阶段的小鼠的生殖发情前期，发情期，发情后期，或动情简单的，非侵入性的收集和阴道涂片细胞学评估。我们还介绍了如何阴道细胞学检查反映循环过渡，通过对小鼠的生殖周期的激素水平相关。

进行阴道灌洗，结晶紫染色法和阴道细胞学评价小鼠发情周期分期鉴定

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

performing-vaginal-lavage-crystal-violet-staining-vaginal-cytological

Research

JoVE Journal

Biology

110.1K 次观看.  Neural Regeneration Laboratory and Ottawa Institute of Systems Biology. 在这里，我们介绍了如何识别阶段的小鼠的生殖发情前期，发情期，发情后期，或动情简单的，非侵入性的收集和阴道涂片细胞学评估。我们还介绍了如何阴道细胞学检查反映循环过渡，通过对小鼠的生殖周期的激素水平相关。

视频: Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

科研

生物学

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

乳腺的发育和功能在小鼠模型的评价

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.

活细胞周期分析<em&gt;果蝇</em&gt;组织使用同调声聚焦流式细胞仪Vybrant的DyeCycle紫DNA染色

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

焦磷酸测序的微生物鉴定和表征

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

精子发生的细胞学分析：Live和固定制剂<em&gt;果蝇</em&gt;睾丸

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

果蝇 guttifera的分期蛹期和机翼色素沉着的测量方法

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

尼罗红与油红 O 染色法测定秀丽线虫的脂质丰度及脂质分布评价

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

各种鼠标脂肪库的识别和解剖

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...