作者想承认4月诡计和 Dr. 罗杰勃洛为协助与动物尸检和腺隔离, 分别。这项工作的经费是由费城骨科医学中心的长期老化疾病的支持。

本议定书中涉及动物主体的程序由费城骨科医学院动物保育和使用委员会审查批准, 所有技术均在严格道德准则. 
 1. 示例采购和处理 <ol> <li> 选择适当的动物主题和地点到 CO 2 会议厅。对于这个实验, 使用8-10 周的老女性产妇 B6。Cg-Cav1tmMIs/J 和 C57BI/6J. </li>
	<li> 打开气体流量到30-40%。一旦动物在大约2分钟以后明显地是无意识的, 打开气体阀门到充分的压力为另外 5 min. 
	注意: 在 CO 2 停止后, 通过观察呼吸的可见迹象 (如胸部的移动), 确认动物死亡的时间为10分钟。如果动物还活着, 它可能被放回 CO 2 室。颈椎脱位也可以使用, 虽然它是不推荐的, 因为血液可能堆积在乳腺周围的解剖和结果的干扰. </li>
	<li> 在牺牲后, 将胴体固定在仰卧位, 并浸透70% 乙醇. </li>
	<li> 用镊子和尼克用小手术剪刀捏住耻骨上方的毛皮。旋转剪刀, 沿腹中线移动的尾部到颅侧切开毛皮. </li>
	<li> 用更大的剪刀或止血, 直截了当地解剖皮下筋膜, 用小心不要刺穿腹膜。沿切口远端两侧的水平边缘切开. </li>
	<li> 将毛皮皮瓣打开, 再用70% 乙醇进行喷雾. 
	注: 虽然使用70% 乙醇允许更好的视觉区分腺体和周围的皮下组织, 小心避免干燥的组织由于使用本解决方案。为了避免干燥, 在一个时间框架内去除腺体不超过4分钟。作为70% 乙醇的替代品, 研究者也可以替代一般的隔离缓冲器, 例如 1x PBS, 以避免组织脱水. </li>
	<li> 定位乳腺的兴趣, 滑动一 #4 手术刀刀片沿皮瓣内侧, 切割乳腺和相关的皮肤脂肪从真皮. 
	注: 3.5x 外科微放大镜可帮助更容易的腺体可视化. </li>
	<li> 立即淹没新孤立的腺体在锥形管包含10:1 溶液-组织容积10% 中性缓冲福尔马林24-48 小时. 
	注意: 根据研究的目的, 许多替代固定可能被使用, 如甲醛, 乙醇用于基因组研究, 以及商业上可用的 RNA 保存缓冲. </li>
	<li> 按照用于石蜡嵌入的机构协议, 将样本提交给供应商进行处理、嵌入和切片/滑动安装。5和 #181 节组织; m. 
	注: 由于脂肪周围腺体过剩, 考虑去除100-200 和 #181; 我的组织前切片和染色. </li>
</ol> 2。组织染色 <ol> <li> 免疫组化 <ol> <li> 放置在58和 #730 上的热块上的幻灯片; C 为1小时融化石蜡. 
		注意: 融化的石蜡可能会滑落。密切监视或在幻灯片下放置擦拭以捕获径流蜡. 
		注意: 此步骤不是必需的, 可以跳过。如果结果不如预期, 则添加此步骤可能会提高结果. </li>
		<li> 在包含100% 二甲苯的滑动罐中孵育幻灯片30分钟, 确保完全浸没。重复一次. 
		注意: 在这一点上, 应进行目视检查, 以确保组织切片不含脂肪组织和石蜡。如果额外的脂肪组织或蜡是明显的, 该幻灯片应 re-submerged 在二甲苯。小心, 尽量减少对二甲苯的暴露, 因为这会导致组织收缩. </li>
		<li> 水化的组织在一个含有100% 乙醇的滑动罐中10分钟重复一次. </li>
		<li> 将幻灯片移动到包含95% 乙醇的滑动罐中10分钟重复一次. </li>
		<li> 将幻灯片移动到包含75% 乙醇的滑动罐中5分钟 </li>
		<li> 将幻灯片移动到包含50% 乙醇的滑动罐中5分钟 </li>
		<li> 在热板或微波中煮沸10毫米柠檬酸钠溶液 (pH 值 6.0), 然后倒入 Coplin 罐. 
		注意: 在加热时要仔细监控解决方案, 并小心避免沸腾。容器会很热。使用保护来避免灼伤. </li>
		<li> 缓慢地将幻灯片放入 Coplin 罐中, 确保完全浸入, 并在100和 #176 孵育; C 为10分钟以检索表位。移除和小心干燥的幻灯片. </li>
		<li> 用疏水标记在组织周围绘制屏障。添加足够的内源酶阻滞剂 (在商业上可用的过氧化氢和叠氮化钠) 覆盖组织并孵育10分钟. </li>
		<li> 在1x 磷酸盐缓冲盐水 (PBS) 中淹没幻灯片10分钟 </li>
		<li> 在 1x PBS 中添加足够的10% 驴血清以覆盖组织并在室温下孵育1小时. 
		注: 实验可以暂停在这里, 储存在湿的室内的幻灯片在4和 #176; C 过夜。当驴血清被发现产生最佳的染色结果时, 研究者可能希望测试不同的血清, 如牛血清白蛋白 (BSA), 山羊血清或马血清, 以确定最佳的结果。如果使用 BSA, 调查人员应在使用前准备此新的. </li>
		<li> 从幻灯片中醒酒多余的阻滞剂, 并在1% 血清中直接添加适当稀释的主抗体, 以确保组织均匀地覆盖在溶液中。在室温下孵育30分钟. 
		注: 此步骤可持续较长时间, 潮湿室贮存于4及 #176; c. 确保在这一步中包括适当的负控制幻灯片 ( 例如 没有主抗体、已知的抗原阴性组织、 等等 的幻灯片). </li>
		<li> 通过流动约1毫升的蝶 2 O 超过组织和孵化 1 1x PBS 5 分钟的变化小心冲洗幻灯片. </li>
		<li> 醒酒过量的 PBS 和添加足够的辣根过氧化物酶标签, 以覆盖组织和孵化30分钟在一个湿润的会议厅. 
		注意: 在这一步, 研究者可能希望使用荧光共轭二级抗体进行荧光染色。如果需要, 则应相应地修改所描述的后续步骤. </li>
		<li> 通过流动约1毫升的蝶 2 O 超过组织和孵化 1 1x PBS 5 分钟的变化小心冲洗幻灯片. </li>
		<li> diaminobenzidine (民建联) 加原解决方案根据制造商建议的比率和涡旋混合. 
		注意: 除了本协议中使用的工具外, 许多 ready-made 套件都是商用的. </li>
		<li> 从幻灯片中醒酒多余的缓冲区, 并将2-3 滴 (10-50 和 #181; L) 的原染色直接添加到组织和在潮湿的室内孵育5分钟。通过在组织上流动大约1毫升蝶 2 O 来仔细冲洗幻灯片. 
		警告: 原是剧毒的。避免与皮肤的污渍直接接触, 并在危险废物中收集流量. </li>
	</ol> </li> <li> 苏木精和曙红 (H & #38; E) <ol> 在原孵化后最后冲洗后, 在哈里斯苏木精中淹没幻灯片 2-2. 5 分钟轻轻冲洗在自来水下的幻灯片1-2 分钟 <li>
		注意: 小心避免直接将水喷到组织上. </li>
		<li> 在差异化解决方案中淹没幻灯片2-3 秒 (0.25 毫升的盐酸在100毫升的70% 乙醇中)。轻轻地在自来水下冲洗幻灯片约1-2 分钟. </li>
		<li> 淹没幻灯片蓝色代理 (4.5 毫克碳酸钙在100毫升自来水, pH 值调整到 9.4) 为六十年代. 三十年代用95% 乙醇冲洗幻灯片. </li>
		<li> 在酒精性曙红 Y 中淹没幻灯片2-3 分钟 </li>
		<li> 脱水组织在2变化95% 乙醇每1分钟. </li>
		<li> 脱水组织在 1 100% 乙醇的变化 1 min. </li>
		<li> 小心地用不起毛的擦干幻灯片。应用1滴 (约 100-200, #181; L) 合成, 非水, 树脂安装介质滑动和应用片. 
		注: 如果选择了不同的染色方法, 则可能需要不同的安装介质。例如, 如果研究者选择了一种荧光共轭的二级抗体, 那么水的安装将更加理想. </li>
		<li> 允许在室温下通宵设置幻灯片. </li>
	</ol> </li> <li> Picrosirius 红色 (PSR) 着色 <ol> <li> 选择要染色的幻灯片, 并通过步骤 6 (50% 乙醇浸泡) 遵循免疫组化协议. </li>
		<li> 在 PSR 染色中淹没幻灯片1小时 </li>
		<li> 淹没幻灯片0.5% 乙酸为1-2 秒, 两次, 以区分污渍. </li>
		<li> 脱水组织在2变化95% 乙醇每1分钟. </li>
		<li> 脱水组织在 1 100% 乙醇的变化 1 min. </li>
		<li> 清除幻灯片, 在100% 二甲苯中短暂地淹没约3-4 秒. </li>
		<li> 小心地用不起毛的擦干幻灯片。应用1滴 (约 100-200, #181; L) 合成, 非水, 树脂安装介质滑动和应用片. </li>
	</ol> </li> </ol> 3。示例分析 <ol> <li> 导管分析 <ol> <li> 选择染色 #945 的幻灯片;-SMA。使用装有摄像机安装物镜的光学显微镜, 在20X 放大倍率下收集具有代表性的图像. </li>
		<li> 使用 ImageJ (NIH), 区分和计数每个图像中的管道。这些可以手动枚举, 或者你可以给单个管道分配一个数值分数。要分配一个数字分数, 打开 ImageJ 并选择插件。下一步选择 "分析", 然后从下拉菜单中选择 "单元格计数器"。单击 "初始化", 然后突出显示类型 1, 开始标记管道。完成后, 选择 "结果" 以查看导管计数. 
		注意: 检查结构是否是导管, 而不是血管. </li>
		<li> in 和 #39; 设置测量和 #39; 选项, 检查和 #39;P erimeter 和 #39; #39; 区域和 #34;. </li>
		<li> 使用手绘多边形工具, 在上皮车厢的每个管道周围绘制一条线 (可见辅助 #945;-SMA 染色)。选择和 #39; 测量和 #39; 并记录外围和区域. </li>
		<li> 再次使用手绘多边形工具, 沿管腔内部的导管上皮顶端绘制一条线。选择和 #39; 测量和 #39; 并记录外围和区域. </li>
		<li> 从上皮室的周长减去腔隔间的周长, 以获得导管上皮的周长。从上皮室的区域减去腔室的面积, 以获得导管上皮的面积. </li>
	</ol> </li> <li> 免疫组化分析 <ol> <li> 将明图像上载到分析软件, 如 ImageJ 或斐济套件. 
		注意: 本协议概述了使用 ImageJ 和 IHC 工具箱插件进行染色分析的步骤. </li>
		<li> 从 "插件" 菜单中打开 "IHC 工具箱"。在和 #34; 选择模型和 #34; 打开的组合框, 选择适当的着色 ( 即 H-民建联, PSR, 等 )。选择 #34; 颜色和 #34; 选项以隔离污渍。这将打开一个结果窗口. 
		注意: 这种方法是适当的, 如果有兴趣的蛋白质位于细胞外或胞浆空间, 或绑定到细胞膜。如果感兴趣的蛋白质是核, 选择和 #34; 原子核和 #34; 将提示插件分析正染色核的图像. </li>
		<li> 使用 "颜色选择器" 幻灯片确保在不包含背景或过多的污点排除的情况下正确隔离污渍. 
		注意: 如果自动模式无法检测到污点或不产生可接受的图像, 请在确定的染色区域上绘制一个正方形的感兴趣区域 (ROI)。在 IHC 工具箱窗口中, 选择和 #34; 训练和 #34; 将插件定向到相应的目标. </li>
		<li> 将图像转换为16位。转到 图像和 #8594; 类型和 #8594; #160; 16 位. </li>
		<li> 阈值图像。转到 图像和 #160; #8594; #160; 调整 #8594; #160; 自动阈值. </li>
		<li> 通过在图像中的刻度栏上直接绘制直线 ROI, 然后分配长度来设置图像测量比例。若要设置缩放栏, 请转到 分析和 #8594; #160; 设置缩放比例. 输入所需长度单位的像素数 ( 例如 每50和 #181 的120像素; m). </li>
		<li> 测量结果区域和平均灰色值。转到 分析和 #8594; #160; 设置度量值 并通过单击 分析和 #8594; 和 #160; 测量 </li> 来收集测量。
	</ol>
	</li>
</ol>

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M., Engler, A. J., Slone, R. D., Galante, L. L., Schwarzbauer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+expression+modulates+mammary+epithelial+cell+proliferation+during+acinar+differentiation.">Fibronectin expression modulates mammary epithelial cell proliferation during acinar differentiation.</a> Cancer Res. 68 (9), 3185-3192 (2008).</li><li>Krause, S., Brock, A., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraductal+injection+for+localized+drug+delivery+to+the+mouse+mammary+gland.">Intraductal injection for localized drug delivery to the mouse mammary gland.</a> J Vis Exp. (80), (2013).</li></ol>

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本文介绍了一种分离完整的小鼠乳腺的技术, 用于下游组织中 ECM 表达和导管形态学的组织学分析。关于导管形态学的分析, 这一方法能够快速研究基于染色组织学切片的导管结构。导管分析的其他方法依赖于染料的注入, 使导管树的可视化, 这些方法在技术上具有挑战性和耗时。
在乳腺癌中, ECM 被报道为异常。1,2,<sup class="xr...

乳腺癌的特点是有相当程度的组织纤维化1,2,3,4。被称为 ECM, 这个泡沫塑料实体是在不同程度的发现在所有组织和主要由一个复杂的网状纤维和 non-fibrillar 胶原蛋白, 弹力素, 和糖蛋白, 除了各种信号分子, 是隔离在这个矩阵。在稳态条件下, ECM 的沉积和降解受到严格控制。5在乳腺肿瘤发生过程中, ECM 沉积和降解的平衡被破坏。因此, 据报道, 乳腺肿瘤表达了丰富的 ECM 蛋白, 如胶原、纤维连接蛋白和素等。6这些蛋白的异常表达除了增加基质交联的模式外, 还记录了促进乳腺肿瘤的进展、转移和治疗的抵抗力1、3、 4,7,8,9。
为了评估 ECM 成分和导管形态学, 对完整乳腺进行了分离。在这里, 我们使用雌性产妇小鼠缺乏 caveolin-1, 一个完整的膜蛋白已被连接到一个积极的乳腺肿瘤签名10,11,12, 并控制女性产妇 B6鼠.这些组织的组织学处理和染色允许对几种 ECM 蛋白的鉴定以及导管形态学的表征。
整体而言, 完整的乳腺隔离使研究人员有机会研究组织范围内的形态学或细胞变化, 以响应外源或内源性因素。这项技术的缺点与2维 (2D) 组织切片的分析相关联, 而不是3D 的角度, 这将使导管树的复杂形态学产生更完整的图像。鉴于乳腺内细胞细胞和细胞 ECM 相互作用的复杂性, 完整、完整的腺体分离有利于有效分析乳腺各区的导管形态学和蛋白质表达腺.

<table>
	<tbody>
		<tr>
			<td>Light Microscope</td>
			<td>Olympus</td>
			<td>BX43</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Camera</td>
			<td>Olympus</td>
			<td>DP73</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td>Olympus</td>
			<td>cellSens Entry software</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>NIH</td>
			<td>ImageJ</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5x-R Surgical Micro Loupes</td>
			<td>Rose Micro Solutions</td>
			<td></td>
			<td>Magnification at researcher&#39;s preference</td>
		</tr>
		<tr>
			<td>Mayo Scissors</td>
			<td>Medline</td>
			<td>DYND04035</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining Rack</td>
			<td>Fisher Scientific</td>
			<td>121</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining Dish</td>
			<td>Fisher Scientific</td>
			<td>112</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin Jars</td>
			<td>Fisher Scientific</td>
			<td>19-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td>Size appropriate for tissue</td>
		</tr>
		<tr>
			<td>IHC EnVision+ Kit (HRP, Mouse, DAB+)</td>
			<td>Dako</td>
			<td>K400611-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrosirius Red Kit</td>
			<td>Abcam</td>
			<td>AB150681</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y, alcoholic</td>
			<td>Sigma-Aldrich</td>
			<td>HT110132</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>HHS16</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>EMD Millipore</td>
			<td>S30</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes, Reagent Grade</td>
			<td>Sigma-Aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 200 proof</td>
			<td>Sigma-Aldrich</td>
			<td>792780</td>
			<td>suitable for molecular biology</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline, 1x</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Fisher Scientific</td>
			<td>S279-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>202932</td>
			<td></td>
		</tr>
		<tr>
			<td>Permanent Mounting Medium</td>
			<td>Dako</td>
			<td>S1964</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt&#39;s Mounting Medium</td>
			<td>Sigma-Aldrich</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin antibody</td>
			<td>Abcam</td>
			<td>AB23750</td>
			<td></td>
		</tr>
		<tr>
			<td>Tenascin-C antibody</td>
			<td>Abcam</td>
			<td>AB108930</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha Smooth Muscle Actin antibody</td>
			<td>Abcam</td>
			<td>AB124964</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Envision Dual Link System HRP</td>
			<td>Dako</td>
			<td>K4065</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of intact, whole mouse mammary glands for analysis of extracellular matrix expression and gland morphology

本程序的目的是从雌性产妇小鼠中获取 #4 腹乳腺, 以评估 ECM 表达和导管结构。在这里, 一个小的口袋下面的皮肤是创建使用梅奥剪刀, 允许分离的腺体内皮下组织的基础腹膜。3.5x 手术微放大镜的使用, 辅助了腺体的可视化。该毛皮被倒置和固定回来, 允许鉴定完整的乳房脂肪垫。每一个 #4 的腹腺都是通过滑动解剖刀刀片在皮下层和腺体之间进行了坦率的解剖。立即收获后, 腺体被放置在10% 中性缓冲福尔马林随后组织处理。切除整个腺体是有利的, 因为它主要消除了风险, 排除重要的组织范围之间的相互作用导管上皮细胞和其他微细胞的人口, 可能会错过的部分活检。该方法的缺点之一是使用固定组织的序列切片, 限制了导管形态发生和蛋白质表达对腺体内离散位置的分析。因此, 3 维 (3D) 的导管结构和蛋白质表达的变化是不容易获得的。总的来说, 该技术适用于需要完整完整的小鼠乳腺进行下游研究, 如发育性导管形态发生或乳腺癌。

雌性小鼠有5对乳腺。具体地说, 有一对宫颈腺体 (#1)、两对胸腔 (#2 和 #3)、一对腹腺 (#4) 和1对腹股沟 (#5) (图 1A)。在这里, 我们隔离了 #4 腺体, 因为它们易于辨认。在某些情况下, #4 和 #5 的腺体被孤立在一起, 因为两者之间的区别是困难的。为了隔离完整的 #4 腹乳腺, 毛皮被固定和小心地从下腹部分离出来, 揭示了由箭头指示的 #4 腺体的?...

Watch this Scientific Journal Video about Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology at JoVE.com

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

在这里, 我们提出了一个完整的, 完整的小鼠乳腺分离的协议, 以研究细胞外基质 (ECM) 的表达和导管形态学。小鼠 #4 腹腺从8-10 周大的雌性产妇小鼠中提取, 固定在中性缓冲福尔马林中, 用免疫组织化学方法对 ECM 蛋白进行切片和染色。

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 ...

isolation-intact-whole-mouse-mammary-glands-for-analysis

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JoVE Journal

Biology

16.1K 次观看.  University of Nebraska Medical Center. 在这里, 我们提出了一个完整的, 完整的小鼠乳腺分离的协议, 以研究细胞外基质 (ECM) 的表达和导管形态学。小鼠 #4 腹腺从8-10 周大的雌性产妇小鼠中提取, 固定在中性缓冲福尔马林中, 用免疫组织化学方法对 ECM 蛋白进行切片和染色。

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

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

切片乳腺全身为病变鉴别

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

科研

癌症研究

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

C57BL/6J小鼠乳腺间质细胞和癌细胞的移植

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

医学

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

通过细胞外囊泡/外来体转移乳腺基质上皮细胞和乳腺腔细胞之间的乳腺形成能力

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

发育生物学

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

蛋白质 - 蛋白质相互作用的分析和鼠乳腺中间隙，紧密和粘附连接部位之间的共定位

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

使用Sholl分析方法量化大鼠乳腺组织中的分支密度

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

通过微分试穿素生成马赛克乳腺有机体

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

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 ...

生物学

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

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 ...