美国国立癌症研究所（U54 CA00100970），美国国立癌症研究所（1 R03 CA150040 - 01），ACS（116602 - PF - 09 - 018 - 01 - CNE）

1。去除腹部的乳腺和准备整装<ol><li>根据IACUC指引安乐死的动物。引脚从它的腿的动物在它的后面，用针或磁带的双腿上表面板，并用乙醇（酒精）擦拭皮肤湿。提起皮肤有点与钳，用锋利的剪刀，开始对＃5乳头和削减对颈部皮肤中线切口。请倒Y切口从中线对的后腿。如果胸部乳腺增生病也将被收集，使＃2乳头对一双前腿之间的中线也是Ÿ切口。 </li><li>解剖的皮肤倒Y切口一侧打开，用剪刀，暴露腹部＃4乳腺，乳腺是附着在皮肤上。引脚针在表面上板的皮肤，完全暴露腺体。工作的时候一边和腺体。 </li><li>剖析乳腺免费使用锋利的剪刀和/或手术刀，从近端面积接近乳头和腺体的末端对工作，对动物的脊椎从皮肤。 （这可能需要几分钟，尤其是在一个旧的大鼠。） </li><li>立即蔓延到一个适当标记的玻片上（使用适当的永久性记号笔和/或“钻石笔”）的分离腺，并蔓延到原来的大小和形状在原地仔细相应的腺体。载玻片必须大于腺体。之后蔓延到幻灯片上的腺体，它坐在桌子上一段时间，使腺体坚持到幻灯片，但不让它干。 </li><li>放入包含一罐卡诺固定液（75％冰乙酸，25％的无水乙醇=乙醇）的幻灯片，让它在室温（RT）固定在2天或更长时间的通风柜。幻灯片也可以留在一个较长时期内的固定液。 </li><li>在室温下1小时，洗净，在70％乙醇的幻灯片。 </li><li>蒸馏水冲洗，在室温下30分钟。 </li><li>染色为2天或更长的时间（直到你看到淋巴结染色的胭脂红铝染色*）;看看背面的幻灯片）。 </li><li>洗涤系列的乙醇，在每1小时：70％ - &gt; 95％ - 100％。最后一次洗涤后，在绝对乙醇在二甲苯中的腺体。让RT通风柜坐至少2天。这最后一步是结算的意义delipidation乳腺，乳腺脂肪垫，并在其后增加透明度。油腻腺体是结算时间是必需的。 </li><li>安装与使用安装的媒体，如Permount，盖单。干的好让幻灯片（数天）前一个立体显微镜下观察。 *胭脂红铝染色： 将1克朱砂（Sigma公司C1022）和2.5克硫酸铝钾（Sigma公司A7167）在500毫升蒸馏水，煮沸20分钟。调整最终体积500毫升水。过滤和冷藏。解决方案可用于几个月。丢弃时颜色变弱。 </li></ol> 2。乳腺整装形态分析乳腺整个坐骑形态是根据以下的终点和乳腺癌的风险相关的结果分析。 <ol><li>乳腺整个PND的21安装（青春期前年龄）： <ul><li>上皮生长： <ul><li>从乳头上皮树（毫米）的距离，用一把尺子测量</li></ul></li><li>恶变的可能性： <ul><li> TEBs（末端芽），光学显微镜下计数。 TEBs是只有在乳腺上皮树的末端位于最大的球根结构</li></ul></li><li>辨证： <ul><li> AB1类（肺泡芽）评分（0-5）。肺泡芽遍布上皮。 </li><li> AB2型得分（0-5） </li></ul></li></ul> 光显微镜下取得Wholes坐骑。从AB1和AB2型的得分值增加为最终分化得分。 </li><li>乳腺整个安装收集的PND 50（后青春期的年龄）： <ul><li>上皮生长： <ul><li>从淋巴结上皮树（毫米）的距离，用一把尺子衡量。 </li><li>从上皮树的一角，脂肪垫的距离，用一把尺子衡量。 </li></ul></li><li>恶变的可能性： <ul><li>数TEBs，光学显微镜下计数</li></ul></li><li>辨证： <ul><li> AB1得分（0-5） </li><li> AB2型得分（0-5） </li><li>小叶得分（0-5） </li></ul></li></ul> 光显微镜下取得Wholes坐骑。分化将评估在两个方面。首先，从AB1，AB2型和小叶的评分值增加最终分化得分。此外，小叶得分和AB1 + AB2得分之间的比例计算。 ABS区分小叶，小叶和ABS之间的比例越高，越有区别的腺体。 </li></ol> 3。触诊大鼠乳腺肿瘤的测量及相关乳腺形态<ol><li>要开始此过程中，按住老鼠，抓在背后的手掌整个身体的头部后面用食指和拇指和相反的腋窝下第二个手指。所以它是躺在它的后路，将大鼠和触诊检测到任何乳腺肿瘤;​​扪及肿瘤应该感到像一个“疙瘩”。 </li><li>接下来，使用一个游标卡尺测量的宽度，长度，高度和肿瘤。计算肿瘤体积，使用椭球体公式，卷=1/6πabc，其中&#39;A&#39;，&#39;B&#39;=宽度=长度，&#39;C&#39;=身高。 </li><li>手工记录肿瘤的位置和大小在每周的笔记本。这些数据将被转移到电子表格后。 </li></ol> 4。代表性的成果乳腺脂肪垫和处理，以wholemount小心剥离将使乳腺的发育状态的评估。当每一步都做得正确，整个乳腺上皮树是清晰可见的脂肪垫内，这使得容易测定TEBs和肺泡芽和小叶的密度计算。在准备wholemounts的失误可能包括解剖，整个脂肪垫，固定不足，和不足，结算失败。 评估将提供乳腺形态结构，目前的数量，可能会引起乳腺肿瘤（TEBs），（肺泡芽和小叶）上皮结构的分化程度，以及增长的措施（导管伸长）的信息。重要的是单独TEBs终端两端的，后者是位于末端的上皮细胞，同样以TEBs，但他们是比TEBs小，不引起恶性肿瘤。也看到终端两端的上皮树内。 <img src="/files/ftp_upload/2260/2260fig1.jpg" alt="图1" /> 图1：代表性的结果做好准备乳腺整装 ，腺体的所有组件已正确解剖和胭脂红染色是最优的。 <img src="/files/ftp_upload/2260/2260fig2.jpg" alt="图2" /> 图2：一个准备不足的乳腺整装代表性的结果，腺体已经正确的解剖（远端部分腺体缺失），一直没有得到很好的拉伸和delipidation还没有完全发生。

<ol>
	<li>Rasmussen, S. B., Young, L. J. T., Smith, G. H., Ip, M. M., Asch, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+mammary+gland+whole+mounts+form+mice.">Preparing mammary gland whole mounts form mice.</a> Methods in Mammary Gland Biology and Breast Cancer Research. , 75-85 (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+statements+from+Mammary+Gland+Whole+Mount+Round+Robin+meeting.">Consensus statements from Mammary Gland Whole Mount Round Robin meeting.</a> , (2009).</li><li>Hilakivi-Clarke, L., Clarke, R., Onojafe, I., Raygada, M., Cho, E., &amp;amp, L. i. p. p. m. a. n., E, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+maternal+diet+high+in+n-6+polyunsaturated+fats+alters+mammary+gland+development,+puberty+onset,+and+breast+cancer+risk+among+female+rat+offspring.">A maternal diet high in n-6 polyunsaturated fats alters mammary gland development, puberty onset, and breast cancer risk among female rat offspring.</a> Proc Natl Acad Sci. 94, 9372-9377 (1997).</li><li>Russo, I. H., Russo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+gland+neoplasia+in+long-term+rodent+studies.">Mammary gland neoplasia in long-term rodent studies.</a> Environ. Health Perspect. 104 (9), 938-967 (1996).</li><li>Hilakivi-Clarke, L., de Assis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+origins+of+breast+cancer.">Fetal origins of breast cancer.</a> TRENDS in Endocrinol and Metabol. 17 (9), 340-348 (2006).</li><li>Hilakivi-Clarke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+modulation+of+terminal+end+buds:+its+relevance+to+breast+cancer+prevention.">Nutritional modulation of terminal end buds: its relevance to breast cancer prevention.</a> Curr Cancer Drug Targets. 7 (4), 465-474 (2007).</li><li>Silva Idos, S., De Stavola, B., Mccormack, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaborative+Group+On+Pre-Natal+Risk+Factors+And+Subsequent+Risk+Of+Breast+Cancer.+Birth+size+and+breast+cancer+risk:+re-analysis+of+individual+participant+data+from+32+studies.">Collaborative Group On Pre-Natal Risk Factors And Subsequent Risk Of Breast Cancer. Birth size and breast cancer risk: re-analysis of individual participant data from 32 studies.</a> PloS Medicine. 5, e193-e193 (2008).</li><li>Warri, A., Saarinen, N. M., Makela, S., Hilakivi-Clarke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+early+life+genistein+exposures+in+modifying+breast+cancer+risk.">The role of early life genistein exposures in modifying breast cancer risk.</a> Br J Cancer. 98, 1485-1493 (2008).</li><li>Russo, J., Hu, Y. F., Yang, X., Russo, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental,+cellular,+and+molecular+basis+of+human+breast+cancer.">Developmental, cellular, and molecular basis of human breast cancer.</a> J Natl Cancer Inst Monogr. , 17-37 (2000).</li><li>Oza, A. M., Boyd, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammographic+parenchymal+patterns:+A+marker+of+breast+cancer+risk.">Mammographic parenchymal patterns: A marker of breast cancer risk.</a> Epidemiological Reviews. 15, 196-208 (1993).</li></ol>

评估乳腺形态的最终点和上皮树的生长，可用于预测是否早期的生活饮食的操作，或在子宫内或青春期的激素环境改变的其他操作，修改后乳腺癌的风险。由于在人类乳腺癌是乳腺结构（终端导管小叶单位，TDLUs）大鼠TEBs相似的启动，这种技术可用于确定早期生活中的接触可能会影响到患乳腺癌的风险。此外，乳腺密度高的妇女增加乳腺癌的4-6倍的风险，并评估乳腺上皮树的生长，可用于识别的决定因素乳腺密度和减少这种密度的因素。要获得此信息，应充分解剖乳腺，适当在玻片上捉襟见肘，因此，它的所有组件可以在显微镜下可视化。此外，适当胭脂红染色和二甲苯delipidation将提供适用于乳腺癌的风险评估的整个坐骑。

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		<div>
		<table border="1">
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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Glacial acetic acid</td>
<td>&nbsp;</td>
<td>EM</td>
<td>AX0073-9</td>
<td>Carnoy's Fixative</td>
</tr>
<tr>
<td>Absolute ethanol</td>
<td>&nbsp;</td>
<td>The Waner-Graham Co.</td>
<td>64-17-5</td>
<td>Carnoy's Fixative</td>
</tr>
<tr>
<td>Carmine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C1022</td>
<td>Carmine solution</td>
</tr>
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<td>Aluminum potassium sulfate</td>
<td>&nbsp;</td>
<td>Sigma</td>
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changes in mammary gland morphology and breast cancer risk in rats

在乳腺癌显示的啮齿类动物模型的研究时， 暴露在子宫内和青春期期间，饮食/荷尔蒙因素对乳腺进行广泛的建模和重新建模，改变致癌物诱发乳腺肿瘤的易感性。类似的研究结果已在人类描述：例如，高出生体重会增加以后患乳腺癌的风险，并在童年的大豆膳食摄入量减少患乳腺癌的风险。据认为，这些产前和产后的饮食修改诱发持久的形态在​​乳腺的变化，反过来说，修改以后的生活中患乳腺癌的风险。这些形态的变化可能反映了表观遗传修饰，如DNA甲基化的变化，组蛋白和miRNA的表达，进而影响基因的转录。在这篇文章中，我们描述了如何在乳腺形态的变化可以预测在大鼠乳腺癌的风险。我们的协议，具体介绍如何解剖和删除大鼠腹部乳腺，以及如何准备乳腺整个坐骑。它还介绍了如何根据三个终点（末端芽数，上皮细胞伸长和分化），并利用这些数据来预测发展乳腺癌的风险分析乳腺形态。

Watch this Scientific Journal Video about Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats at JoVE.com

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

我们的协议描述了如何解剖大鼠腹部的乳腺和如何准备乳腺整个坐骑。它还介绍了如何分析乳腺形态，使用三个端点（末端芽数，上皮细胞伸长和分化），并利用这些结果来预测膳食修改暴露大鼠乳腺癌的风险<em&gt;在子宫内</em&gt;或期间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 ...

changes-in-mammary-gland-morphology-and-breast-cancer-risk-in-rats

Research

JoVE Journal

Biology

29.3K 次观看.  Georgetown University. 我们的协议描述了如何解剖大鼠腹部的乳腺和如何准备乳腺整个坐骑。它还介绍了如何分析乳腺形态，使用三个端点（末端芽数，上皮细胞伸长和分化），并利用这些结果来预测膳食修改暴露大鼠乳腺癌的风险在子宫内或期间prepuberty。

视频: Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

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 ...

科研

生物学

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

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 ...

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 ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

的三种不同方法对乳腺癌细胞株确定细胞增殖的比较

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

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 ...

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, ...

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 ...