这些研究是由CIHR CBCRA补助金，以DWL。知识产权是由CIHR - STP，FRSQ和CIHR奖学金资助。 MKGS是由OGS和CIHR - STP奖学金。作者感谢协助他用鼠标育种凯文巴尔。

1。乳腺淋巴清扫术<ol><li>安乐死的鼠标使用CO 2吸入。如果可能，避免颈椎脱位，在颈部和乳腺周围的血液中积累的结果，因为它可能会损害大血管，使剥离更加困难。但是，如果其他条件，在相同的鼠标，如催产素的血药浓度，评估，替代安乐死方法可能是必要的，尽管这必须由您IACUC评估。 </li><li>在铝箔包裹的聚苯乙烯泡沫塑料，在它的后面放置鼠标和蔓延四肢。引脚牢固四肢使用16至20号针头（图1A）。 </li><li>鼠标慷慨地喷用70％乙醇。 </li><li>使用镊子，拉起在中线的腹部皮肤，使一个小切口锋利的剪刀。 </li><li>从切口，切开皮肤，动物的颈部，同时避免穿刺腹腔或胸腔（图1B）。 </li><li>切前肢正中切口的皮肤上，导致在一个"Y形"（图1B）。削减后方四肢皮肤相同的方式（图1B）。 </li><li>使用止血钳，轻轻剥离鼠标侧面的腹部和胸部的皮肤。如果需要，轻轻切开结膜组织。 </li><li>修复皮肤使用16至20表针，揭露附着到皮肤下面的乳腺聚苯乙烯泡沫塑料。进行和完成时间（图1C）一方。 </li><li>使用镊子，轻轻乳腺的利益和减少之间的腺体和皮肤用锋利的小剪刀，开始从外部对脊柱的动物。确保削减仔细，所以没有皮肤或肌肉，保持腺体。所有乳腺增生病（图1）可以使用此协议被删除，但腹部（＃4）乳腺是最佳的整体安装。 第二和第三腺体一些叉的肌肉，虽然它们可以被用于整个安装和组织学，他们可能不适合于所有的分析。 </li></ol> 2。整装<ol><li>至少一天前清扫，准备1G的胭脂红和2.5G的硫酸铝钾溶解在500毫升蒸馏水煮沸20分钟1升锥形瓶中胭脂红明矾溶液。调整最终体积500毫升水。筛选通过的Whatman纸，并添加少量的麝香草酚（几粒）作为防腐剂。冷藏。胭脂红明矾溶液染色后，可以倒出股票瓶，多次重复使用几个星期。颜色变得暗淡时，作出新的解决方案。 </li><li>鼠标（步骤1）乳腺切除后，直接在玻片上传播。尝试尽可能扁平化，使原来在原地形状。正确拉伸时，腺体会很好地坚持在幻灯片上。 </li><li>通风柜在一个罐子在室温4小时修复，在幻灯片上，沉浸在卡诺固定液（100％乙醇，氯仿，冰醋酸; 6:3:1）的腺体。请注意，其他人已经用了2小时，这似乎是足够的。此外，腺体也可以固定在4 ° C过夜。 </li><li> 70％的乙醇洗为15分钟。然后，补充水分，水逐渐从JAR中删除一半的乙醇溶液和取代DDH 2 O孵育5分钟。重复3次。另外，使用70％ - 乙醇和水的浴缸，每次5分钟 - 35％ - 15％。 </li><li>在蒸馏水冲洗5分钟。 </li><li>在室温下染色胭脂红明矾过夜。应该指出，染色可能需要更长的时间取决于腺体厚度。 </li><li>卸下胭脂红明矾染色重用，并逐步通过串行乙醇浴（50％，70％，95％，100％）脱水染色组织5分钟每二甲苯明确，或在一个无毒的替代，如组织明确，过夜。脱水和结算可能还需要较长的孵化较厚的样品。 </li><li>沉浸乳腺水杨酸甲酯。乳腺增生病，可以保持在通风橱中的甲基水杨酸，直到图像准备采取。 </li><li>图像可以在通风柜定位在三角架上（使用"宏"功能，如果有的话），放置一盏灯表的幻灯片，一个普通的数字相机。使用解剖显微镜高倍率。另外，乳腺增生病，可以安装在如Permount媒体（2.7步）后，允许采取任何成像平台提供的坐骑图片以外的化学通风柜足够薄。 </li><li>为了量化乳腺发育，使用整个安装，可以评估的后果（或分行）沿管道部分，主管的长度，或上皮脂肪组织的面积比等测量。最后，经过摄影documen仓储乳腺整装可以嵌入石蜡切片和常规病理染色。 </li></ol> 3。催产素诱导的牛奶弹射<ol><li>分开后，他们被美联储大坝的幼崽。进行检测前，请等待1个小时。 </li><li>牺牲的母亲并没有消除他们从动物，暴露前（步骤1.1至1.8）所述的乳腺。 </li><li>开始均匀地下降，直接对利息乳腺PBS或催产素的解决方案，使用移液管的检测。可用于大范围的剂量;〜8 pg / mL的生理剂量通常用于。然而，非生理性的高剂量（1毫克/毫升），可以使用，以确保在转基因小鼠模型的情况下的响应。对于此过程，它是建议用胸（＃2-3）腺体，作为对照（暴露，以PBS）和对方的一个侧面测试牛奶射血分数（催产素曝光）。 </li><li>孵育1分钟，删除的解决方案，仔细用移液管吸。牛奶进入管道的入口可以监控录制的录像带，或服用前后PBS或催产素曝光的图像。 </li></ol> 4。代表性的成果: 在一个良好的整装，是很好的传播和乳腺上皮细胞的管道很容易观察到的（图2A）。如果腺体是没有得到很好的传播，它可以从幻灯片抬起放在固定液的一部分或全部。腺体就会变得硬而无法使用（图2B）。同样，如果没有很好地解剖腺，无论是一些上皮细胞的一部分可以失踪（图2C）或剩余部分的皮肤或腹部肌肉会干扰分析与腺体（图2D）。 对于催产素的检测，重要的是要分开大坝相同数量的时间对每一个场合哺乳仔兔。这样，您将确保没有牛奶或少量的牛奶是在管道催产素暴露之前中，也有足够的牛奶是可以弹出导管肌上皮收缩（图3A）在肺泡。如果幼崽过早地被删除，牛奶可以累积在肺泡和管道渲染催产素的效果难以监控（图3B）。另外，如果去除后小狗还为时过早实验，牛奶会不会有时间来积聚在肺泡和催产素诱导的肌上皮细胞收缩，不会触发到足够的牛奶被观察的管道（图3C）释放。 <img src="/files/ftp_upload/2828/2828fig1.jpg" alt="图1"/> 图1。小鼠乳腺清扫。在它的后面，四肢（一）引脚鼠标。剪下从腹部到颈部的皮肤。腹部中心，每个肢体虚线（二）所示，然后从垂直切口。一侧动物的皮肤上，轻轻地揭下针，露出了乳腺增生病（三）。 <img src="/files/ftp_upload/2828/2828fig2.jpg" alt="图2"/> 图2小鼠乳腺整个坐骑。上皮管（箭头）和淋巴结（箭头），很容易观察到一个很好的传播乳腺腺体全挂载＃4（一）。不善传播的乳腺有可能成为脱离幻灯片和折叠，使其无法使用（二）。不当切除的乳腺可以有缺少的部分（C）或肌肉或皮肤件，其余（四）。请注意第2次和3路腺体叉的肌肉，但仍然可以用于整个安装或组织学。 <img src="/files/ftp_upload/2828/2828fig3.jpg" alt="图3"/> 图3。催产素诱导牛奶弹射，可观察到上皮管（箭头），催产素曝光（一）。不过，如果管道已经在去年哺乳后的幼崽，进一步积累牛奶在管道接触后，催产素（箭头）的检测，由于不适当的长期延迟开始与牛奶（箭头）充满很难（二）观察。另外，短的潜伏期后，小狗的哺乳不会让牛奶足够的积累后，催产素曝光（三）妥善管（箭头）观察到肺泡。前和后叶催产素使用数字相机的曝光（CYBERSHOT，索尼）图像。 <img src="/files/ftp_upload/2828/2828fig4.jpg" alt="图4"/> 图4。整个安装和催产素治疗实验的示意图表示。

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乳腺发育和功能的紧密协调。突变小鼠中可能隐藏的基因突变，会损害乳腺的发育和功能，突出了需要评估腺架构13,12。本文中描述的乳腺发育的各个阶段，整个安装可以执行。通常情况下，上皮细胞的发展是评估在青春期开始（〜4周龄），青春期（〜6周龄）中，进入青春期后（约10-12周龄）2,10 。在这几个阶段，管分支的数量，可确定的末端芽和管道的长度以量化程度的发育14,15。同样，alveologenesis（肺泡怀孕期间的发展）通常在2-3点确定，在怀孕期间，在整个装入腺制剂16，发生在哺乳期和复旧的结构变化也可以被观察和量化。然而，从后期的孕妇和哺乳期的小鼠整体坐骑量化可以是困难的，由于组织的密度。这是建议，结合病理分析整装，尤其是对于那些阶段12 ，。更详细的组织学评价，乳腺可以去掉，使用相同的协议，在这份报告中指出，在福尔马林固定，并以苏木精和曙红染色。整装也可以石蜡包埋和组织学用一次成像已执行。对于这两种技术，适当的和微妙的乳腺的解剖是至关重要的。如果突变小鼠正在评估，重要的是尽可能最接近的对照组小鼠比较这些老鼠，因为不同的小鼠品系，可以显示变量的副作用或三级后果 ，或alveologenesis 17。例如，BALB / c小鼠相比，C57BL / 6小鼠表现出减少副作用分支和alveologenesis 17,18 。因此，BALB / c小鼠，不应被用来作为在C57BL / 6小鼠的背景下产生的转基因小鼠的控制。虽然整个安装提供了总值发育缺陷的洞察力，乳腺的功能，可能还需要使用其他补充技术进行评估。 乳腺的主要职能是生产和交付牛奶幼仔。这些过程不仅需要在怀孕期间分泌管腔细胞的正常分化，但还协调和高效的收缩，周围的肺泡， 沿运奶8导管的肌上皮细胞。虽然牛奶产量和质量可以由牛奶中的成分分析评估12评估，除其他外，幼 ​​崽的送奶受损的原因是难以评估。事实上，牛奶新生幼崽的肚子观察到的情况下，可以链接到许多缺陷，如牛奶产量受损，无法哺乳幼崽的母亲或牛奶弹射缺陷的行为缺陷。从肺泡进入管道评价牛奶的运输和弹射缺陷的方法之一，是诱发肌上皮收缩，使用催产素，如本文中所述。然而，牛奶的运输和射血分数量化是困难的，因为体内催产素的作用是快速的（通常是牛奶，可以在暴露后1分钟内观察到的管道），即使使用低生理剂量催产素10。因此，在牛奶两个鼠标线之间的弹射效率略有差异可能不能使用这种技术观察。量化这种反应是很难的，但一个半定量评估催产素诱导牛奶弹射的方式，是研究各种不同的剂量下的反应，并确定治疗所需的时间，以填补与乳管。 。在所有情况下，等待周期之间的最后乳猪检测开始小狗的一致性是至关重要的。对于哺乳期或牛奶弹射缺陷识别更准确的量化，这种方法可以由牛奶蛋白和信号通路，如β-酪蛋白和STAT5，分别量化补充， 或在体外肌上皮细胞收缩后，催产素曝光 19 。 综上所述，转基因小鼠模型为研究乳腺功能和发展是重要的，广泛使用，并评估在这些过程中的关键基因的作用。乳腺上皮细胞的结构可以很容易地观察到整个装载在发展的所有阶段使用的正确切除腺体。虽然哺乳仔兔营养不良导致的缺陷，可以有很多的起源，在牛奶弹射的不足，可以评估催产素诱导同步收缩肌上皮细胞原位。总之，这两个技术（图4）可以给特定的基因突变的效果的重要见解tions或乳腺发育和功能上的缺失。

<table border="1"><tbody><tr><th>试剂</th><th>公司</th><th>目录编号</th></tr><tr><td>胭脂红</td><td> Sigma - Aldrich公司</td><td> C1022 </td></tr><tr><td>硫酸铝钾</td><td> Sigma - Aldrich公司</td><td> A6435 </td></tr><tr><td>百里</td><td> Sigma - Aldrich公司</td><td> T0501 </td></tr><tr><td>水杨酸甲酯</td><td> Sigma - Aldrich公司</td><td> M6752 </td></tr><tr><td>催产素</td><td> Sigma - Aldrich公司</td><td> O3251 </td></tr></tbody></table>

evaluation of mammary gland development and function in mouse models

15-20叶组成的人体乳腺分泌乳汁，到乳头管系统开放的一个分支。这些叶是自己的终末导管小叶分泌肺泡和融合的管道1单位组成。在小鼠实验中，观察到类似的架构在怀孕管道和肺泡内的结缔组织基质中穿插。小鼠乳腺上皮细胞是像由两层细胞，管腔细胞基底膜2的范围表示的肌上皮细胞的外层包围的内层组成的导管系统树。出生时，只有残留的导管树目前，主风管和15-20分支机构组成。伸长率和放大分公司开始在青春期的开始，4周龄左右，3,4,5激素的影响下。在10个星期，大部分基质入侵的管道由一个复杂的系统，将发生在每个发情周期，直到怀孕 2分支和回归周期。在怀孕开始，开始第二阶段的发展，与上皮细胞的增殖和分化形成葡萄状的牛奶分泌结构称为肺泡 6,7的。整个分娩和哺乳，牛奶是生产管腔分泌细胞，并储存在肺泡腔内。催产素的释放，仔兔哺乳引起的神经反射刺激，诱导肺泡周围和沿导管的肌上皮细胞的同步收缩，让牛奶通过管道输送到乳头它成为可用的幼崽 8 。乳腺的发育，分化和功能的紧密配合和要求，不仅基质和上皮细胞之间的相互作用，而且之间的肌上皮细胞和管腔内的上皮细胞9,10,11。从而，在这些相互作用中有牵连的许多基因的突变可能会损害或者在妊娠晚期和分泌的激活 ，导致泌乳12,13在青春期或肺泡在怀孕早期，分化形成导管伸长。在这篇文章中，我们描述了如何解剖小鼠乳腺和评估他们的发展，使用整个坐骑。我们还演示了如何评价肌上皮收缩和牛奶弹射使用前体内催产素的功能测定。对乳腺的发育和功能的基因突变的影响，因此可以在原地决定执行这两种技术在突变体和野生型对照组小鼠。

Watch this Scientific Journal Video about Evaluation of Mammary Gland Development and Function in Mouse Models at JoVE.com

Evaluation of Mammary Gland Development and Function in Mouse Models

这种方法介绍如何剖析和评估小鼠乳腺发育和功能。切除乳腺评估使用全挂载的发展程度，而牛奶弹射使用催产素为基础的肌上皮细胞收缩测定评价。

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 ...

evaluation-of-mammary-gland-development-and-function-in-mouse-models

Nanyang Technological University

Research

JoVE Journal

Biology

37.1K 次观看.  University of Western Ontario. 这种方法介绍如何剖析和评估小鼠乳腺发育和功能。切除乳腺评估使用全挂载的发展程度，而牛奶弹射使用催产素为基础的肌上皮细胞收缩测定评价。

视频: Evaluation of Mammary Gland Development and Function in Mouse Models

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

使用IVIS频谱的乳腺肿瘤在体内生物发光成像

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

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 ...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

变化在大鼠乳腺形态和患乳腺癌的风险

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

鼠标涎腺干细胞的分离

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

精子冷冻保存和恢复使用I ·冷冻包

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

导管内注射局部给药的小鼠乳腺

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

斑马鱼肾功能评价采用荧光测定间隙

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

对小鼠乳腺冰冻切片间接免疫荧光

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

完整、全小鼠乳腺的分离及细胞外基质表达及腺体形态学分析

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...