这项工作是支持由美国国防部（BC075554 BG和BC061403到DK），美国国立卫生研究院（U54GM064346到杰威尔; CA100324赛马，JES和JW; CA77522的JES，U54CA126511 JC和BG）。我们感谢Entenberg D.显微镜，M.成像盒和J.波拉德（阿尔伯特爱因斯坦医学院）F4/80抗体制造的帮助Rottenkolber帮助。

1。荧光肿瘤生成肿瘤细胞注射到乳腺脂肪垫： <ol><li>长出的细胞系表达Dendra2蛋白（细胞质标记）40 - 80％汇合。 </li><li>冲洗菜至少3次，用PBS W / O 型的Ca 2 +或Mg 2 + 。 </li><li>添加每10厘米的菜，并培育3毫升的胰蛋白酶在37℃直至大部分细胞分离，然后撞到一个平面上动摇它的菜。 </li><li>冲洗关闭菜中的所有细胞，并用刮板（橡胶警察），以及收集矩阵。新增的5毫升的PBS。取一个分装在离心计数。 </li><li>离心5分钟，在800克。 </li><li>吸用PBS重悬，以浓度为5 - 10倍10 6 /毫升。冰商店，直到注射（30 min内注入）。 </li><li>无菌罩内含有4-5周龄女性免疫（如免疫缺陷）缺陷小鼠放在一个笼子里。 </li><li>第四届（腹部）用70％乙醇的乳头周围喷洒的面积。 </li><li>乳腺脂肪垫注入0.1毫升。如果可以找到一个助手，一个人就可以举行到位的鼠标，而其他针插入乳腺脂肪垫内。如果你自己注射用异氟醚，浅麻醉下鼠标。 </li><li>一旦肿瘤已经发展到5-​​7毫米，乳腺影像窗口（MIW）应插入。 注：另外，对于某些应用，可以插入MIW健康的乳腺脂肪垫的顶部和细胞随后被注入。这种方法允许在生理环境中瞬时转染的细胞成像。 </li></ol> 2。手工制作的乳腺影像窗口（MIW）： <ol><li>组织级塑料MIWs（图1A），以保证生物相容性。对于手动建设中，我们使用5年或10厘米的菜。 </li><li>把乳胶手套。 </li><li>热了起来，推着一个圆形硬物，对加热盘（我们使用一个1英寸的dremmel位）一个组织培养皿中，共创一个曲面。 </li><li>切出的菜使用了激烈的刀片中心。 </li><li>使用一个小，圆锥状dremmel位，使中心的圆顶形的塑料底座（直径6 - 7MM）一个洞。 </li><li>塑料底座的边缘打磨dremmel进一步提交的顺利。 </li><li>文件一个平坦的表面玻璃盖玻片的塑料基地顶部。应提交的，平坦的表面直径9 - 10mm的。 </li><li>胶8毫米的圆形玻璃盖玻片上平展的表面使用的强力胶（1号）（氰基丙烯酸酯胶粘剂）。 </li><li>等待胶水干（15分钟）。 </li><li>使用了激烈的26G针八个缝合穿刺孔从外部（玻璃）基地内。孔应均匀分布各地的盖玻片，盖玻片边缘0.5 - 1mm的距离。 </li><li>拓宽使用5-0缝合针的孔。 </li><li>在塑料基穿刺孔的内表面凹凸不平。用沙纸，使这个表面完全平滑的。 </li><li>更改小塑料颗粒，用小刷子MIW的手套和刷子。 </li><li>用去离子水清洗MIW。 </li><li>使用70％乙醇洗净MIW。使用棉条的清洁，所以它是完全透明的玻璃。如果有云里雾里的点，从目前的胶水蒸汽，小心使用棉条丙酮。 </li><li>在每边为3 h的紫外线照射消毒MIW。 </li></ol>半手工制作的乳腺影像窗口（MIW）： 为了制造塑料的基础，我们目前使用的硅橡胶铸模创建使用手工制作的MIWs。模具是由两个部分硅橡胶，使双方的正面和背面的原始充满聚酯树脂，然后立即联手的精确副本。液体聚酯树脂混合在一起，在硬盘的结构完全治愈在48小时时9:10和结果。塑料是没有打破或随着时间的推移成为黄色的紫外线保护和档案。 基地治愈后，步骤2.8-2.16都做同样的方式为手动MIWs。 3。插入的MIW： <ol><li> 4 个乳头周围的区域应该有一个小的（5-7毫米直径）肿瘤取决于细胞类型（图1B）的肿瘤细胞注射后的数天/周。已经过去了多少时间，Dendra2 photoswitchable蛋白或其他荧光蛋白表达的细胞注射后取决于所使用的细胞线。肿瘤不应明显坏死，应该有一个完整的皮肤肿瘤上方的头发。肿瘤坏死的小鼠应安乐死。 </li><li>准备手术的无菌空间，放下一块无菌罩内的无菌布。把一块无菌克auze在中间，并奠定了几MIWs和它周围的无菌文书：我们使用标准的小剪刀，弹簧剪刀，microdissecting镊子，microdissecting钳，持针器。保持无菌的Q -提示，一瓶70％的乙醇和罩在角落的无菌手套。 </li><li>鼠标是在无菌罩麻醉使用的HBSS 2.5％avertin IP注射液（20μL/ G），或另外10微克/公斤氯胺酮+ 10微克/克甲苯噻嗪（审批和订购，联系你的动物保健研究所）。 注：应准备Avertin解决方案双周，过滤消毒使用0.22微米的过滤器和储存500μL分装在4 ° C（在黑暗中）。 </li><li>取出头发剃上述使用一个小动物剃须刀的肿瘤面积。 </li><li>删除其余的头发使用奈尔脱毛膏（可在任何药店）。清洁皮肤使用乙醇浸的Q -提示。 </li><li>动物转移到无菌纱布和眼药膏眼睛，以保持干燥和感染。 </li><li>用优碘消毒皮肤，并用70％乙醇清洗。 </li><li>转移到无菌罩内的无菌手术布的动物。 </li><li>拉扯皮肤，立即使用产钳和切割〜2mm的切口，在皮肤乳头内侧。 </li><li>分开使用夹层的剪刀和镊子皮肤底层的乳腺脂肪垫。皮肤在这种分离的步骤大小，将容纳插入MIW洞里。 </li><li>如果意外的船只是在sugery打，使用无菌的Q -技巧，以消除任何生产/停止出血，成为过度的血液。 </li><li>有皮肤到位MIW基地和缝合上使用非吸收线和反向切割针插入MIW。 </li><li>使用组织粘合剂或氰基丙烯酸酯，以填补在缝合孔和保护皮肤的MIW。 </li><li> TMP - SMX抗生素混合添加：（磺胺甲基异恶唑0.6毫克/毫升，甲氧0.12毫克/毫升）入笼前和手术后3天一瓶水。 </li><li>在麻醉状态下的动物保持avertin腹腔注射后1 - 4H。由于手术通常需要〜45分钟，重要的是要帮助动物恢复之后： </li><li>将手加热垫（37 ° C）在笼子的底部（在药店），并用纱布覆盖。 </li><li>保持垫之上的动物，它的肚子上，直到它关闭，并开始步行。 </li><li>如果动物是无意识3h以上，注入0.3毫升的HBSS腹腔补液。 </li><li>动物是可以恢复在未来3-4天前发生的第一次成像会议。 </li></ol> 4。成像盒建设保证MIW是坐在平面以上的显微镜物镜，成像盒。它也有利于通过控制温度和麻醉异氟醚的恒定气流。这个盒子是由有机玻璃粘在一起使用塑料焊接。它配备的具体显微镜阶段，因此其底部的形状各不相同，取决​​于使用（图2显示了徕卡SP5显微镜载物台装有框的计划）显微镜。 一盒以厘米为单位的尺寸为L = 11.4厘米，宽7.6厘米，H = 4.4厘米（图2A）。盒子的底部由空心板，12.7x8.4厘米大小，里面适合徕卡SP5显微镜载物台，和两个滑动“门”，5.7 × 5.7 cm_each，从而形成一个圆形开口（D = 2.22厘米）时关闭。 MIW适合这样的开场白。前片含麻醉分娩的入口孔，而侧块包含一个电源插座孔，导致真空。 需要注意的是冷凝器和幻灯片持有人需要拆除成像盒放置前。 5。成像盒使用<ol><li>与异氟醚麻醉下放置动物的眼睛和应用眼药膏。 </li><li>将异氟醚麻醉机废气成像盒正面，对外面的进水孔（图2B）。 </li><li>将第二个管成像盒的插座孔。此管应连接到气体过滤器，然后在真空。 </li><li>打开盒盖，打开箱门和地方的动物，MIW面朝底部，和成箱的滑动车门。 </li><li>按住方块和调整的底部，使MIW基地举行它们之间的固定门。 MIW基地应在同一级别的成像盒门，而MIW盖玻片应低于这个水平只是一个几毫米。请确保MIW之间的推拉门底部的定位，确保盖玻片（玻璃部分MIW）平躺，平行于底部的门框。这将确保正确聚焦。 </li><li>降低显微镜物镜，允许框的位置空间在显微镜阶段的顶部。 </li><li>放在显微镜舞台上的方块，调整的阶段，因此，MIW盖玻片上述目标。 </li><li>聚焦的目标和启动成像。 </li><li>虽然使用异氟醚，动物应监测呼吸频率的目视检查。这可以在视觉上，或通过监测呼吸文物成像集合（这些干扰与数据采集）期间出现的频率。 MouseOx脉搏血氧仪系统（斯塔尔生命科学公司）已成功使用。这样的数据可以收集不断无需打开房间的灯检查动物。 注：每个成像会后应便利包装的小手，在加热垫纱布或kimwipes置于它在笼子里的动物，动物恢复。 注意：如果浸泡的目的是通过MIW形象，应该清除浸泡介质（甘油，水，油）的成像窗口。 MIW应检查成像过程中可能发生的任何裂纹或其它损坏。这是多种成像会议期间收集的数据的关键。 </li></ol> 6。 Photoswitching和Dendra2标记细胞成像程序是徕卡SP5的共聚焦显微镜;不同焦平面的激光功率，可用的目标和软件选项使用其他共聚焦显微镜，多光子成像成立，因此，下文所述的协议应为指导用于优化您的显微镜实验。 <ol><li>通过观察肿瘤与绿色荧光过滤器的目镜（10倍），找到主要血管，这是明显的流动和铺设在同一焦平面单位。 </li><li>位置的领域，并切换到光电倍增管检测中心的船只之一。 </li><li>设置两个通道的顺序成像收集。的渠道之一，使用488nm激光线（10％的电力），并从细胞外基质（480 - 495nm）和绿表Dendra2（505 - 540nm处）的排放收集的散射。第二条通道，使用543nm激光线（90％的电力）和收集Dendra2红色形式（555 - 600nm处）的排放量。收集预photoswitching的三维图像。 </li><li>使用的投资回报率的扫描选项，光控选择一个使用405纳米的激光线（30％的电力，20-40扫描）的细胞人口。收集排放的蛋白质的红色形式监测开关。 </li><li>使用相同的顺序6.3常规，收集后切换3D图像。 </li><li>如果photoswitching一个以上的地区，在肿瘤，应采取在绿色和红色通道（图3A，B），通过使用数码相机的目镜的照片。这将有助于方向，在随后的成像会。 注：此外，血管可以暂时采用尾静脉注射荧光葡聚糖（级联蓝色或的Alexa Fluor 647，既10kDa）标记。右旋糖酐注射后几个小时，将离开血管和永久标签的巨噬细胞。 </li></ol>代表性的成果： 图3C显示了一个长方形photoswitched的区域（红色）面向正交相的血管（无荧光）。非photoswitched细胞是绿色的，而从细胞外基质的散射是紫色的。 Image是一个四象沿Z轴（20-50微米深度）的最大强度投影。

<ol>
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<td>PBS w/o Ca, Mg</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14190-144</td>
<td>&nbsp;</td>
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<td>Trypsin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25300</td>
<td>&nbsp;</td>
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<td>HBSS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14025</td>
<td>&nbsp;</td>
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<td>DMEM media (MDA-MB-231 cells)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11965</td>
<td>&nbsp;</td>
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<td>Isoflurane(Aerrane)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td># NDC 10019-773-40</td>
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<td>SCID mouse</td>
<td>&nbsp;</td>
<td>NCI</td>
<td>N/A</td>
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<td>Circular coverslip 8 mm</td>
<td>&nbsp;</td>
<td>Scientific, Inc.</td>
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dendra2 photoswitching through the mammary imaging window

在过去的十年，活体显微镜的分辨率在单细胞在小鼠和大鼠乳腺肿瘤<sup&gt; 1-4</sup&gt;成如肿瘤细胞迁移，侵袭和血管内的转移行为的机制导致了重要的见解<sup&gt; 5，6</sup&gt;，血管生成<sup&gt; 3</sup&gt;和免疫细胞的反应<sup&gt; 7-9</sup&gt;。最近，我们报道了多个活体成像会议的一种技术活老鼠形象原位乳腺癌<sup&gt; 10</sup&gt;。对于这一点，我们已经开发出乳腺影像窗口（MIW）和优化的成像参数为Dendra2<sup&gt; 11</sup&gt; photoswitching和成像<em&gt;在体内</em&gt;。在这里，我们描述MIW，插入肿瘤成像的Dendra2标记的肿瘤细胞，使用一个定制的成像盒的顶部MIW制造业的协议。该协议可用于不同的微环境在肿瘤中的肿瘤细胞的转移行为形象和长期的血管，肿瘤细胞与宿主细胞的长期成像。

Watch this Scientific Journal Video about Dendra2 Photoswitching through the Mammary Imaging Window at JoVE.com

Dendra2 Photoswitching through the Mammary Imaging Window

Dendra2标记通过乳腺成像窗口肿瘤细胞的活体photoswitching和跟踪是一种技术，它使我们的图像多天的时间刻度选择了肿瘤微环境中的肿瘤细胞的转移行为。

通过乳腺成像窗口Dendra2 Photoswitching

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4  has resulted in important insights into ...

dendra2-photoswitching-through-the-mammary-imaging-window

Research

JoVE Journal

Biology

11.6K 次观看.  Albert Einstein College of Medicine - Yeshiva University. Dendra2标记通过乳腺成像窗口肿瘤细胞的活体photoswitching和跟踪是一种技术，它使我们的图像多天的时间刻度选择了肿瘤微环境中的肿瘤细胞的转移行为。

视频: Dendra2 Photoswitching through the Mammary Imaging Window

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

通过颅窗口在新皮层的血流量为双光子成像方法

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

科研

生物学

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 ...

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

小鼠乳腺上皮细胞形成过程中Lactogenic分化的微球体

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

乳腺上皮移植手术

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

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 ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

通过影像学的蛹壳<em&gt;果蝇</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.

牛乳腺活检技术

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, ...

In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2

Proteins are synthesized and degraded constantly within a cell to maintain homeostasis. Being able to monitor the degradation of a protein of interest is key to understanding not only its life cycle, but also to uncover imbalances in the proteostasis network. This method shows how to track the degradation of the disease-causing protein huntingtin. Two versions of huntingtin fused to Dendra2 are expressed in the C. elegans nervous system: a physiological version or one with an expanded and pathogenic stretch of glutamines. Dendra2 is a photoconvertible fluorescent protein; upon a short ultraviolet (UV) irradiation pulse, Dendra2 switches its excitation/emission spectra from green to red. Similar to a pulse-chase experiment, the turnover of the converted red-Dendra2 can be monitored and quantified, regardless of the interference from newly synthesized green-Dendra2. Using confocal-based microscopy and due to the optical transparency of C. elegans, it is possible to monitor and quantify the degradation of huntingtin-Dendra2 in a living, aging organism. Neuronal huntingtin-Dendra2 is partially degraded soon after conversion and cleared further over time. The systems controlling degradation are deficient in the presence of mutant huntingtin and are further impaired with aging. Neuronal subtypes within the same nervous system exhibit different turnover capacities for huntingtin-Dendra2. Overall, monitoring any protein of interest fused to Dendra2 can provide important information not only on its degradation and the players of the proteostasis network involved, but also on its location, trafficking, and transport.

In Vivo Quantification of Protein Turnover in Aging C. Elegans using Photoconvertible Dendra2

Presented here is a protocol to monitor degradation of the protein huntingtin fused to the photoconvertible fluorophore Dendra2.

使用光可转换的登德拉2，在老化的蛋白质周转量中，在体内C.Elegans

Proteins are synthesized and degraded constantly within a cell to maintain homeostasis. Being able to monitor the degradation of a protein of interest is ...

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

使用带有可更换盖子的乳腺成像窗口进行纵向活体腔内显微镜检查

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in ...

Intravital Subcellular Microscopy of the Mammary Gland

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676&#160;or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

<meta charset="utf8"></head><body>乳腺活体亚细胞显微镜检查

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

乳腺活体亚细胞显微镜检查

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...