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>

作者没有什么可透露的。

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。1,2,6,9具体来说, ECM 丰度和/或交联模式的变化已被报道影响肿瘤的进展, 转移和治疗阻力3,4,6,7 ,8,9。此外, 还报告了 ECM 环境的变化, 改变导管上皮细胞的形态和增殖17, 有可能导致一种有利于肿瘤萌生的情况。当我们利用一个 caveolin-1 缺陷动物模型来评估 ECM 表达的变化和导管形态学的损失后, caveolin-1, 你可以应用这种方法在任何鼠标模型。为了评估 ECM 表达模式和导管体系结构, 我们描述了一种分离完整的乳腺, 用于下游组织学分析的技术。这一方法的结果产生了对 ECM 表达和腺形态学的稳健分析。我们发现, 分析 ECM 表达, 特别是纤维连接蛋白, 从切片和染色前去除100-200 µm 表面组织的部分, 产生了更完整的图像腺纤维化。此外, 我们发现, 使用驴血清, 而不是 BSA 取得更好的质量组织学样本, 其中非特异性染色大大减少。
对于导管结构的分析, 重要的是要使用的部分, 其中组织是完整的, 并包含管道, 其中破损点, 由于不适当的组织处理和切片的结果, 是不明显的。此外, 重要的是要正确区分导管分支, 另一个参数, 一个研究者可能希望测量, 和血管结构, 可以很容易地识别他们单层内皮和嗜酸性结构内腔内除染色外 CD31。在各种 ECM 蛋白染色的切片上观察导管, 上皮标记物αSMA 和 #38; e counterstaining, 突出了导管的细胞成分, 对导管和任何相关分支的计数可通过 H 和 #38 实现;单独染色组织切片。为了更详细地分析导管的形态发生或对 pre-neoplastic 和/或肿瘤病变的存在进行分析, 研究者可能希望利用共聚焦成像来评估活体前体组织。在这种情况下, 相同的技术, 整个腺体切除可以应用与固定和组织处理步骤消除。
本议定书的关键步骤包括使用外科微放大镜, 它不仅能精确识别 #4 腹腺, 而且还允许从上覆盖的真皮和下腹膜中解剖腺体。大量的皮下组织和肌肉的排斥减少了脂肪相关的染色和组织学上的干扰。此外, 为了更好地识别 ECM 在腺体中的表达变化, 我们发现去除100-200 µm 的组织块是有用的, 这有助于消除残留的脂肪组织。虽然全乳腺隔离提供了有关基质蛋白表达和导管形态发生的组织范围变化的信息, 但所描述的技术仅限于从固定的石蜡包埋组织中进行序列切片分析。为了确定导管分支发生变化的程度, 就必须对活体、前体组织进行全贴装成像。在这里, 一个调查员可以使用动词氧合酶来设计定制的, 有条件的荧光报告, 以研究腺体特定的蛋白质相互作用或利用染料照亮导管树之前, 成像18。除此之外, 对整个腺体感兴趣的蛋白质的进一步分析将需要使用西方杂交或质谱或其他高通量技术, 如交联沉淀 (夹子) 或染色质沉淀 (芯片)。在这种情况下, 整个腺体将被解剖, 切成小块, 并相应处理的下游分析。此外, 你也可以在组织切片上使用荧光成像。这项技术对于分析同一组织中的多种蛋白质和蛋白质表达共存是非常理想的。
总之, 我们描述了一种技术, 提供了完整的, 完整的乳腺快速隔离, 并提供了进一步细节的组织学处理和导管分析。该技术的未来应用可以研究肿瘤动物体内导管形态学的 ECM 表达和相关变化。此外, 分析了胶原的取向和纤维直径, 可能有助于分析组织密度的信息, 也可以在 PSR 染色切片上进行。

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 腺体的位置 (图 1B)。#4 腺体被仔细切除, 并检查是否存在任何残留的皮下或肌肉组织。虽然这些在我们的孤立腺体中是不可识别的 (图 1C), 但我们确实注意到, 在切片之前, 组织块中有大量的皮纤维粘连蛋白腺体的皮下组织 (例如:表面) (图 1D) 与腺体100-200 µm 组织块中的组织在切片前被移除 (如深) (图 1e)。为了更好地了解组织纤维化在乳腺的反应外源或内源性因素, 研究者可能希望比较纤维化的表面与深切腺体。对于其他的分析, 它可能是有利的研究人员将组织块的一半, 然后进行组织切片和染色, 如果大量的皮下和/或肌肉组织是存在的腺体解剖。
在隔离后, 腺体被固定在10% 中性缓冲福尔马林中, 经加工和切片染色。在图 2A中描述了除了其他感兴趣的蛋白质外, 用于分析胶原蛋白表达的步骤。在图 2B中显示了 ECM 蛋白胶原、纤维连接蛋白和素 C 的代表性样品。每个图像中的箭头显示这些组织中的导管的存在 (图 2B)。
为了量化导管的形态学特征, 对组织学切片进行治疗是很重要的, 因为处理不当可能导致受损的组织和导管无法测量。不当处理的例子可能是在组织中直接运行缓冲器或水, 在不适当的温度下使用缓冲器, 或者在苛刻的缓冲液中 (如柠檬酸钠和醋酸) 中留下组织样本, 比议定书中规定的时间更长。在图 3a中显示组织损伤发生的组织部分的示例。在这里, 对组织的广泛损伤可以很容易地被观察作为破损点在管道 (图 3A)。这样的截面不能用于导管结构的分析。一个完整的组织标本的例子, 其中的管道是可测量和可量化的显示在图 3B。在这里, 导管不仅是周围的纤维连接蛋白染色的基质, 但也有特点是存在的上皮细胞密集的填充管腔的管道 (图 3B)。重要的是要注意, 在发展过程中, 小鼠乳腺包含一个单一的导管在出生时, 侧副分支发芽, 形成一个更复杂的导管树, 经历了一系列的分支和回归与每个动情周期13. 虽然在这些实验中我们没有考虑到动物的发情周期, 但研究者可能希望这样做, 以便获得独立于动情周期的导管数的信息。研究人员通过 Caligioni14来引用一个协议, 在这种情况下, 可以明显地确定动情周期的阶段。关于定量的导管, 在这些组织学切片中存在多个导管结构并不表示有多个独特的导管, 而是预示了导管生长所产生的更发达的导管树。要列举导管, 你可以使用像 ImageJ (NIH) 这样的图像分析软件为组织切片中的单个导管分配一个数字。研究者可能希望列举导管作为导管数的改变, 这可能是发育异常或病理状况的指示。关于我们的研究, 我们相信导管的结果的增加的数量, 是与 caveolin-1 的损失相关的, 可能驱动生长因子的异常的表示, 例如胰岛素生长因子 1 (IGF-1), 一个已知的老鼠导管的斡旋者增长15.
除了完整的导管存在外, 还有几个导管包含分支, 由纤维连接蛋白染色部分的箭头指示 (图 3C)。为了进行这种分析, 这些导管分支不应被视为单独的导管结构。在评价和测量导管时, 区分导管和血管结构是很重要的。血管结构不仅可以通过存在的内皮单层内衬的流明, 但也存在的核, 嗜酸性结构指示红细胞 (红细胞) 在流明 (图 3D)。虽然在流明内存在嗜酸性的结构表明红细胞和建议的血管, 在腺体收集期间, 红细胞在整个组织切片的位移是可能的。用内皮标记 CD31 确定血管结构是可取的。显示了带有 CD31 的组织学切片的示例 (图 3D)。为了量化导管周长和面积, ImageJ (NIH) 的向量可以被指定为轮廓上皮边界 (周长) 和腔边界的α-SMA 染色组织切片。α-SMA 是一种平滑的肌肉细胞的标志物, 它在上皮细胞中被用作线导管。在显示的图像中, 红线显示上皮边框, 而蓝线则显示腔边框 (图 3E)。在示意图中所示的基质和腔间室之间的区域是由总基质组织和导管上皮细胞 (图 3E) 占据的区域。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56512/56512fig1.jpg"> 
图 1: 分离完整的乳腺, 进行组织学分析.(A)在动物献祭之后, 毛皮被小心地取下, 并被钉回, 露出位于真皮和腹膜之间的乳腺。图示显示颈椎 (#1)、胸椎 (2 和 3)、腹部 (#4) 和腹股沟 (#5) 腺体的相对位置。(B)在解剖动物中显示的 #4 腹腺, 并由红色箭头表示, 是孤立的。(C)典型的 #4 产妇雌性 B6 鼠的腹腺。(D)从腺体中的大量皮下组织, 在切片前未从石蜡块中除去组织 (如表面), 在 ECM 蛋白纤维连接素染色后明显可见。(E) Considerab更少的皮下组织和更多的纤维粘连蛋白染色的基质组织观察从腺体, 其中100-200 µm 的组织已从石蜡切片之前, 切片 (例如深)。组织学切片来自有代表性的样本。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56512/56512fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56512/56512fig2.jpg"> 
图 2:组织学处理和染色.(A)用于组织学处理的步骤的示意图表示。用不同的步骤来分析胶原蛋白的表达。(B)来自雌性产妇 caveolin-1 缺陷动物腺体的 ECM 蛋白胶原、纤维连接蛋白和素的代表性图像。用于胶原蛋白、纤维连接蛋白和素的箭头表示导管的致密基质衬里。PSR: Picrosirius 红。Fn: 纤维连接蛋白。十-c: 素<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56512/56512fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56512/56512fig3.jpg"> 
图 3:导管结构的分析(a)纤维连接蛋白染色切片说明了广泛的组织损伤。这是在导管结构 (箭头) 中出现断裂的标志。(B)一个纤维连接蛋白染色部分说明了完整的导管结构的存在, 这是由一层或多层上皮细胞和周围的基质所包围的流明所标示的。箭头表示个别导管结构。(C)一个纤维连接蛋白染色图像突出显示导管分支的存在, 这是用箭头表示的。注意这些的连续性与大导管结构的流明。(D)血管结构 (红色箭头), 其特征是单层细胞和存在有核的嗜酸性细胞结构, 指示红细胞, 在靠近导管 (黑色箭头) 时经常观察。在左侧的图像中显示低放大倍数, 右侧显示相应的高倍放大图像。这些图像是用 H 和 #38 染色的; E 和素。CD31 被用作血管结构内皮细胞的阳性标记物。显示的是一个大的血管结构, 其中 CD31 阳性细胞可见衬里的血管腔。在左侧的图像中显示低放大倍数, 右侧显示相应的高倍放大图像。(E)导管圆周在α-SMA 染色的图像中测量, 使用矢量 (以红色显示) 来勾勒上皮间隔, 而导管区则使用矢量 (蓝色显示) 来勾勒腔间隔。在间质和腔之间的区域被归类为导管区。刻度栏 = 50 µm. 导管结构示意图, 突出了测量周长和面积的区域。十-c: 素图 E 中的组织学部分被修改了: 汤普森 C et al.16 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56512/56512fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Universidad San Sebastian

Research

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

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

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

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

变化在大鼠乳腺形态和患乳腺癌的风险

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

科研

生物学

Isolation of Mouse Salivary Gland Stem Cells

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

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

鼠标涎腺干细胞的分离

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

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 ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

小鼠气管基底细胞和粘膜下腺体的导管细胞分离

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

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 ...

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

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 also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the 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 ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators 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 ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

一种快速，为隔离，功能可扩展的研究方法，以及细胞衍生的细胞外基质的分析

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

小鼠原发性肾管上皮细胞的分离、鉴定及高通量的细胞外流量分析

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...