The authors would like to thank the Biomarker Imaging&#160;Research Laboratory (BIRL) at Sunnybrook Research Institute for their histology services. Support for this work was provided by the Terry Fox Foundation, the Canadian Breast Cancer Foundation&#8208;the Prairie&#8208;NWT as well as a CIHR grant, #MOP-97996.

1。标本<ol><li>切除乳腺手术从野生型CDH1以及IGFBP7缺失小鼠3天发病后泌乳。 </li><li>传播腺体到玻片上，以帮助恢复本地乳腺形态。 </li></ol> 2，固定和组织处理<ol><li>修正了在中性缓冲4％PFA O / N的乳腺在4℃。 </li><li>存储腺体在70％乙醇前，组织处理。 </li><li>转让腺体小组织处理磁带。 </li><li>使用自动组织脱水处理的组织<ol><li>脱水组织中增加70％乙醇的乙醇和二甲苯浴45分钟，2次95％乙醇45分钟，3次，在100％乙醇中1小时，2倍于二甲苯中45分钟。 </li><li>渗透到组织中与施加压力的真空石蜡3次，每次1小时。 </li></ol></li><li>嵌入在石蜡中的组织以形成块为切片。 </li></ol> 3，组织学和Blockface成像<ol><li>用旋转切片机，直到多余的石蜡除去修剪的石蜡块。 </li><li>使用立式铣床钻1毫米孔的石蜡块中的至少两个角垂直于盒中。 </li><li>安装在所述旋转切片机将组织块。 </li><li>设置在切片前的blockface成像系统19。 </li><li>切片前拍摄的光blockface形象。 </li><li>切四节丝带在在切片机5微米的厚度。 <ol><li>传输带的凉水澡。 </li><li>分隔功能区的第二和第四部分，并挂载它们在显微镜玻片上。选择第二和第四部分提供部分之间有5微米的间隙。 </li><li>展开每个部分在一个温水浴（48℃）来流露它，然后重新将其安装在显微镜幻灯片。 注:Cutting，安装，unwrinkling的章节会导致一些扭曲的部分，如撕裂，褶皱，收缩和扩张。这些文物复杂的组织切片进行登记。 </li><li>使用自动染色机染色用H＆E的章节。 </li><li>使用自动盖片盖玻片的幻灯片。 </li><li>使用数字病理幻灯片扫描仪在感兴趣的分辨率数字化幻灯片。对于这个协议的放大倍数是20倍，分辨率为0.47微米。 </li><li>下采样组织学图像blockface图像，18微米的分辨率。 </li></ol></li></ol> 4，图像配准<ol><li>图像分割和点的选择<ol><li>在blockface图像测量的定位孔的像素值，并使用平均值作为固定阈值来分割在石蜡块的角上的定位孔。 </li><li>由于一些额外的部分可能也被分割使用固定阈值，则使用圆形和分段对象找到孔和丢弃多余的物体的面积。要做到这一点，写一个小代码，并找到（4πX面积）/（周长）2的分割对象的比例。该比率为圆形物体是1。 </li><li>对于每个乳腺中，选择一个blockface图像作为参考，并通过使用定位孔和基于地标的配准技术的中心对齐blockface图像到参考的其余部分。 </li><li>用于对准blockface图象手动段或从背景中提取组织。用最庞大的物体在遮罩协议的其余部分。 </li><li>对于H＆E部分按照下面的步骤进行自动分割。 <ol><li>利用大津阈值技术20分割图像从背景中并创建组织学图像的二值掩码。 </li><li>识别和使用h每个掩码中选择最可观的对象istogram标记的对象。 </li><li>提取这两种组织学和blockface掩盖了一个像素宽的边界点。 </li><li>利用链码算法21，通过分段线性序列来表示边界点拟合。 </li></ol></li></ol></li><li>最初的刚性配准<ol><li>使用傅立叶描述算法22，找到组织学的边界点及其相应的blockface图像之间的初始刚性变换。这最初的变换包括平移，旋转和缩放因子。 </li><li>变换的各组织学图像与初始变换来自之前的步骤获得的。 </li></ol></li><li>刚性注册的细化<ol><li>使用一个滚动的球过滤器23的组织学轮廓去除高曲率边缘部分。 </li><li>选择使用随机均匀分布，其余组织学边界点500点。 </li><li>变换的组织学随机边界点与傅立叶描述所获得的初始转化。 </li><li>选择整套blockface边界点，并使用迭代最近点（ICP）算法24找到blockface边界点，目的地和组织学随机边界点之间的刚体变换。 </li><li>变换来自前面步骤中获得的组织学对准的图像和对齐的组织学图像的堆叠产生的组织学量。 </li><li>使用三维可视化软件创建的组织学卷的视觉形象。 </li></ol></li><li>在5倍的放大倍率观看图像的堆栈<ol><li>下试样原始组织学图像放大5倍。 </li><li>裁剪所述感兴趣区域中的组织学图像中的一个。 </li><li>计算该区域的位置，在使用来自两个步骤登记的刚性变换的组合其他5倍的组织学图像。 </li><li>裁剪的regi感兴趣的所有其他组织学图像大小相同区域的元件。 </li><li>最后细化手动区域之间的对准。编写一个程序，覆盖两幅图像，并允许选择的旋转和在另一个图像中的一个翻译的值，然后保存，当对准被接受的变换后的图像。 </li><li>查看对准5X组织学区的堆栈使用三维可视化软件。 </li></ol></li></ol>

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The authors have nothing to disclose

在这项研究中，我们开发了一种图像配准工作流程，从串行2D组织学图像，它不要求在组织内的内部随机选择的地标或植入的基准标记，这可能会扭曲组织重建三维组织学量。通过所描述的方法，光学blockface图像本身被用作前切片的参考图像。我们用钻在石蜡块外部孔，以帮助对齐blockface图像和纠正在镜头前的石蜡块的2D横向运动。二维组织学图像是相一致的相应的二维图像blockface防止配准误差的...

IGFBP7（胰岛素样生长因子结合蛋白7）是胰岛素样生长因子结合蛋白家族的一个成员，并且已经显示出结合的IGF-1受体4。下调的IGFBP7已知与不良预后乳腺癌5相关联，而IGFBP7在异种移植肿瘤模型中重新引入大大抑制生长6的肿瘤通过诱导细胞凋亡和细胞衰老7。为了研究IGFPB7的影响，一个IGFBP7空小鼠建立5（未发表资料）。虽然这些小鼠不发展的肿瘤，它们显示在卵巢，肌肉和肝脏的组织学变化，以及在乳腺发育模式（未发表数据）的缺陷。有缺陷的表型是首次显示为空的小鼠具有较小的窝产仔数和都无法维持多个大型窝（未发表资料）。 三维组织学卷有潜力提供有用了信息离子进行定量和比较分析病理结果在体积医学图像和评估。三维共聚焦，双光子显微镜可以在本地范围14提供的腺体高分辨率细胞形态的信息，但它具有的视图和深度的有限字段。组织学容积重建提供了更大的空间范围的更多信息。使用传统方法的组织切片，如收缩，膨胀，眼泪和褶皱的制备过程中的一些失真是始料不及的。这些扭曲使得很难寄存器串行组织学图像转换成3D堆叠来重构一个三维体积。作为连续部分与缺陷的数量增加了完整的部分之间的相似性被降低，从而使登记过程更加复杂。 不同的方法已经被提出来注册的组织切片，并建立一个连续的组织学沃lume明。一些技术依赖强度变化8，和其他人是根据第9的形状。对于一些样本的解剖结构，可作为地标10,11沿着与基于地标的登记方法12,13。但这些内部结构可能不会在整个体积和一些标本没有可靠的解剖结构可以识别被检测出。有些团体已经使用成对的注册方法，并连续登记的组织学图像一个到另一个使用轮廓或解剖结构16-18。注册序列组织切片彼此不使用参考图像可以传播准误差和改变组织学量的实际形状。成对登记方法依赖于对组织学切片和内部结构在整个图像的堆叠的形状的一致性;因此，它需要密集采样的标本，其可能并不总是可能的， 例如，对临床标本。 在这条管线，我们使用blockface图像作为一组用于组织学容积重建19参考图像。 Blockface图像被安装在切片机后采取的石蜡组织块，并且每个部分被切断之前。因而，损害个体的连续切片切不干扰的连续切片8,11,15登记。我们捕捉blockface图像与其他群体不同的方式。光学块面部图像是由一个远心透镜光学以消除或尽量减少桶和透视失真，这通常是使用在光学透镜经常发生时获得的。这是在其他公布的方式，使用常规镜头的执行blockface成像所提出的方法的优势之一。的图像被拍摄时的轻微倾斜的角度要使用该块的表面的反射的做卷烟之间的对比度增强UE和石蜡表面并消除深度的组织的阴影中，石蜡的表面之下。一种照相过滤器还用于偏振来自块表面与组织平衡对比度19的光。为了校正在旋转切片机块的位移，二至三孔加工成块的角上，这是很容易检测的blockface图像。这些孔的形心连同基于地标的刚性配准用于对齐blockface图像。

<table border="0" cellpadding="0" cellspacing="0" width="852">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">16% PFA</td>
			<td style="width:179px;">VWR International</td>
			<td style="width:119px;">15710</td>
			<td style="width:295px;">16% Paraformaldehyde solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Small tissue processing cassettes</td>
			<td style="width:179px;">VWR International</td>
			<td>CA95029-956</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:260px;">Leica ASP300 Automated Tissue processor</td>
			<td style="width:179px;">Leica</td>
			<td>14047643515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">100% ethanol</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">S25307B</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Xylene</td>
			<td>VWR International&#160;</td>
			<td>CA95057-822</td>
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			<td height="21" style="height:21px;width:260px;">Paraffin&#160;</td>
			<td style="width:179px;">Thermo Fisher</td>
			<td style="width:119px;">39501006</td>
			<td>Paraplast Tissue Embedding Medium</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Leica EG 1160 Embedding Centre</td>
			<td style="width:179px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Leica rotary microtome</td>
			<td style="width:179px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Milling machine</td>
			<td style="width:179px;">Argo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Microscope slides</td>
			<td style="width:179px;">VWR International&#160;</td>
			<td>CA48312-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">H&#38;E stain</td>
			<td style="width:179px;">VWR International</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Automatic stainer</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Coverslips&#160;</td>
			<td style="width:179px;">VWR International&#160;</td>
			<td>48404-452</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">MEDITE RCM 7000 Glass Coverslipper</td>
			<td style="width:179px;">MEDITE</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Leica SCN400 slide scanner</td>
			<td style="width:179px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">MATLAB</td>
			<td style="width:179px;">MathWorks Inc</td>
			<td>MATLAB 2007b</td>
			<td>Development software</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:260px;">MeVisLab</td>
			<td style="width:179px;">MeVis Medical Solutions AG</td>
			<td>MeVisLab 2.1</td>
			<td>3D visualization software</td>
		</tr>
	</tbody>
</table>

reconstruction of 3-dimensional histology volume and its application to study mouse mammary glands

组织学容积重建有利于三维造型和器官在由细胞组成的宏观水平体积变化的研究。它也可以用于研究和验证在容积医学成像和治疗的新技术和算法。创建不同器官1,2,3的3D高解析度地图集是组织学容积重建的另一种应用。这提供了用于研究组织结构和各种细胞特征之间的空间关系的资源。我们提出了组织学容积重建，它使用一组光学blockface图像的图像配准方法。重构的组织学量表示已处理标本无殉后处理配准误差的可靠的形状。苏木精和曙红（H＆E）染色的两种小鼠乳腺切片使用从海关提取边界点登记到相应的blockface图片在组织学和blockface图像试样的GES。登记的准确性目视评价。乳腺的宏观结构的取向也被肉眼评价在高的分辨率。 本研究界定的不同步骤这个图像配准的管道，从乳腺切除术，通过对三维组织学容积重建。虽然2D组织学图像显示对部分之间的结构差异，组织学三维体积提供了可视化的形状和乳腺的体积差异的能力。

传统的显微镜技术的缺陷是一个器官在微观层面的理解是在一个时间限制在一个现​​场的视图。即使是"完全披露"的幻灯片，它提供了整个滑动部分，不能提供三维信息。随着整个幻灯片，动态扫描技术，我们看到它的一个整体部分的能力有所提高，发展却推断结构需要组织学3D容积重建。 为了更好地表征IGFBP7空鼠标的不足，对腺体切除泌乳期后3天开始进行了三维重建乳腺...

Watch this Scientific Journal Video about Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands at JoVE.com

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

3维组织学音量和其研究小鼠乳腺腺体的应用改造

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

reconstruction-3-dimensional-histology-volume-its-application-to

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.3K 次观看.  University of Toronto. We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands. 

视频: Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

梯级步行任务：一个评分系统，其实际应用。

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

在斑马鱼和镜头移植在眼突变分析中的应用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

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 ...

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 ...

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 ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

维甲酸的应用获取从初级小鼠成骨细胞骨细胞培养

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

肿瘤细胞球体的高通量图像分析:一个用户友好的软件应用来测量球体的大小自动准确

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

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 ...