国家环境健康科学研究所 (NIEHS) 动物护理和使用委员会 (ACUC) 根据 NIEHS 动物研究提案 IIDL 05-46 批准了这项研究的所有方法。
1. 激光显微切割的最佳组织采集
<ol><li>获得2到3月大的老鼠, 男性或女性 C57BL/6。弄死与 CO2的最小值为1分钟或直到呼吸停止。将动物从笼子中取出, 并通过颈椎脱位、斩首或开胸手术来保证死亡。</li>
	<li>解剖前, 确保所有解剖工具清洁和消毒。 注: 为去除眼睛弯曲的剪刀和钳与锯齿状尖端 (首选尖端尺寸: 0.5 x 0.4 毫米) 将需要。</li>
	<li>将鼠标放在其侧面的平坦表面上, 使眼睛容易接近。用镊子抓住眼角 (眼角), 用钳子稳定鼠标, 然后用弯剪刀, 面对曲线远离眼睛, 用眼窝切开眼睛以引导, 直到眼球可以很容易地从插座上取走 (使用曲线不是 剪刀的提示)。</li>
	<li>轻轻拉, 并使用弯曲剪刀, 切断视神经, 从眼眶插座释放眼睛。小心地从眼部清除非眼部组织, 并在1x 磷酸盐缓冲生理盐水 (PBS) 冲洗。对侧眼重复 1.3-1.4。</li>
	<li>在嵌入前, 用纸巾擦拭眼睛以去除水分。将眼睛放入标本模 (25 x 20 x 5 毫米3), 使透镜和视神经与工作台顶平行, 以获得矢状部分。嵌入眼睛在最佳的切削温度 (O.C.T.) 化合物和地方在干燥冰结冰。冷冻后, 将区块存放在摄氏-80 摄氏度。</li>
</ol>2. 激光显微切割用冷冻切片的制备
<ol><li>使用前在紫外线交联器上孵化聚乙烯对苯二甲酸乙二醇酯 (PET) 膜, 最大功率为45分钟。</li>
	<li>在刷子、镊子、联轴器罐和安装卡盘上喷涂 RNase 净化液。彻底擦拭。用无核酸酶的水冲洗, 晾干。用100% 乙醇擦拭恒温器, 避免交叉污染。</li>
	<li>调整恒温器到-18 摄氏度。一次从-80 °c 冰箱中取出一个冰冻块, 用干冰运送到恒温器。允许冻结块平衡恒温器的温度最小为10分钟。</li>
	<li>将少量 O.C.T. 挤压到安装块 (图 1A) 上, 然后从模具中取出组织块, 并小心地将块放在安装卡盘上 (图 1B)。一旦安装卡盘上的组织块被冻结, 插入到恒温器的标本持有者 (图2A)。</li>
	<li>调整恒温器切割8µm 节, 并仔细切片通过 O.C.T. 块, 以达到组织样本。一旦组织被观察, 停止切片, 修剪所有两侧的冰冻块使用干净的剃刀刀片和留下 3-4 毫米的 O.C.T. 周围的组织, 以使处理与油漆刷 (图 2B)。使用新的清洁油漆刷代替辊板之间的每个样品变化, 以避免样品交叉污染。</li>
	<li>部分通过眼睛快速工作。在六十年代用一步的苏木精和伊红 (H & E) 染色液冲洗每第三节, 用自来水漂洗, 空气干燥, 清除剂清洁, 并装在带有盖玻片的带电玻璃滑梯上。查看显微镜下, 继续切片, 直到 TM 是明显的切割部分。</li>
	<li>一旦 TM 开始出现, 切割2节, 并安装在一个带电的玻璃幻灯片上例行的 H & E 染色, 形成一个地图, 以 LCM 切割。 注: 有关地图和染色详细信息, 请参阅第3节。</li>
	<li>在第一个 H & E 地图幻灯片后, 在恒温器中剪切并排成6个串行8µm 厚的部分, 同时安装到 PET 膜幻灯片上 (图3A,B)。通过简单地滑动戴手套拇指沿膜的背面坚持组织。安装后, 立即将 PET 膜滑入干燥冰上的滑动盒中。 注: 可装入的节数较少, 但建议一次将多个截面安装到幻灯片上, 以通过限制每节暴露在室温空气中的时间来减少 RNA 的降解。</li>
	<li>继续部分的眼睛, 每两个 PET 膜幻灯片每6节, 收集两个部分, 在一个带电的玻璃幻灯片, 以创建另一个地图幻灯片的 H & E 染色。继续切片, 大约100系列8µm 厚的部分, 直到 TM 不再可见。确认通过快速染色一节的带电玻璃幻灯片 (见 2.6)。</li>
	<li>将所有 PET 膜幻灯片存放在-80 摄氏度, 供以后 LCM 使用。</li>
</ol>3. H & E 地图滑动染色协议及形态学评价
<ol><li>要可视化并查看带电玻璃幻灯片上的地图幻灯片, 请立即将组织转移到填充有100毫升快速固定溶液的染色罐中, 7 秒. 将其浸入蒸馏水中 10-20 次, 然后将滑块转移到带有干净蒸馏水的耦合罐中。</li>
	<li>从水中取出滑块, 用纸巾擦拭干边。吸管200µL 改性过滤哈里斯苏木精到每个部分和孵化三十年代在室温下。小心冲洗在自来水下的滑动, 直到水运行清楚。将幻灯片的末尾与每个步骤之间的纸巾擦除 (每个步骤之间的污点从 3.2-3.5) 去除多余的液体。</li>
	<li>三十年代在室温下将幻灯片放在 1x PBS 中。然后, 仔细冲洗的幻灯片下运行自来水三十年代。</li>
	<li>将幻灯片 3-4 倍浸入95% 乙醇浴中。应用足够的伊红 Y 染料溶液覆盖在幻灯片上的组织和孵化十五年代在室温下。</li>
	<li>将95% 乙醇浴中的滑块冲洗两次, 每次 10-20 快速浸洗。将100% 乙醇浴中的滑块冲洗两次, 每次 10-20 快速浸洗。然后, 在二甲苯浴中冲洗两次滑动 (10-20 快速浸出每个)。</li>
	<li>通过应用树脂安装介质到组织, 添加盖玻片小心, 以避免气泡, 让它干燥在遮光罩过夜。</li>
	<li>在视觉上或使用数字幻灯片扫描仪查看 H & E 幻灯片。识别具有 TM 清晰可见和可用于切割的地图幻灯片。使用的 PET 膜幻灯片之间的这些地图幻灯片为 LCM。</li>
</ol>4. 聚对苯二甲酸乙二醇酯 (PET) 膜滑动处理和染色协议
<ol><li>在从冷冻机中取出 PET 滑块前, 先用20毫升75% 乙醇溶解0.3 克甲酚紫乙酸酯, 然后放到振动筛上, 用2小时甲酚紫罗兰溶液. 用0.22 个µm 无菌过滤装置过滤溶液, 贮存在4摄氏度, 不超过6星期一秒.</li>
	<li>在1:4 的比例下, 用新鲜的75% 乙醇制备出含酒精溶液。在冰上清洁、无核酸酶、50毫升的锥形聚丙烯管中制备固定 (75% 乙醇) 和脱水溶液 (75%、95%、& 100% 乙醇)。在每个溶液中添加 RNase 抑制剂。</li>
	<li>从-80 °c 冰箱中取出含有冰冻组织切片的滑动盒, 放在干冰上。一次处理一张幻灯片。在三十年代将幻灯片立即放入冷75% 乙醇中, 固定幻灯片。</li>
	<li>轻轻地蘸在核酸酶水 10-十五年代的幻灯片, 以溶解 O.C.T. 而不干扰组织部分。</li>
	<li>小心吸管300µL 1.5% 甲酚紫乙酸溶液直接到切片和孵化四十五年代在室温下。然后, 洗一次, 把它放入75% 乙醇浴为三十年代. 吸管200µL, 在切片上孵育 3-5 s。</li>
	<li>脱水组织通过放置后, 随后在75% 乙醇, 95% 乙醇, 最后, 100% 乙醇浴每三十年代。在每一个步骤之间清除多余的液体, 用纸巾擦拭幻灯片的末端。让滑动空气完全干燥的引擎盖下5分钟。一旦干燥, 立即执行 LCM。 注: 建议避免使用二甲苯的这一步骤, 它可能导致组织切片不适当的粘结到膜滑动和降低组织捕获的效率。</li>
</ol>5. 激光显微切割用 UV 激光
<ol><li>打开计算机并允许启动。打开显微镜白色光源电源。打开紫外线激光键和黄色 LED 将照亮。启动 LCM 软件, 让它充分加载, 现场视频将显示。按下控制器盒上的激光按钮, 绿色指示灯将亮起。</li>
	<li>将新的玻璃滑块装入显微镜的舞台上, 然后将 PET 膜滑动与染色的组织侧直接朝下, 在玻璃滑梯上。确保薄膜和玻璃滑块在显微镜阶段紧密配对。</li>
	<li>首先设置幻灯片限制, 启动 LCM。在目标面板中选择最低的4X 放大倍数 (图 4)。然后通过移动显微镜 xy 阶段来定义薄膜滑动的工作区域, 直到在现场视频提要中可见金属和膜满足的左上角, 按下 "极限 1" 按钮, 在路线图上会出现一个红色矩形。接下来, 移动到 xy 阶段到相反的角落, 并按下 "极限 2" 按钮。按 "扫描" 按钮, 创建一个路线图图像的膜和组织之间的设置限制。</li>
	<li>双击路线图中某个眼睛部分的 tm 附近, tm 可以位于眼前房角的顶点附近 (图5A)。一旦识别出 tm, 将放大倍数提高到 10X, 并手动将焦点放在 tm 上。重复20X 和40X 目标。当 TM 聚焦于40X 目标 (图 5B) 时, 导航到附近的空白区域 (焦点可能需要稍微调整), 选择 "手绘绘图工具", 并在空白的组织区域上绘制一条长线。</li>
	<li>设置激光参数, 使 "剪切速度" 为 34%, "焦点" 为 58%, "电源" 为 70% (图 4), 然后按 "剪切" 按钮。对速度、焦点和功率参数进行精细的激光调整, 直到观察到清晰而精细的切割线 (请参见图 5C)。为精确切割, 确保切割动作遵循绘制线。</li>
	<li>将隔离管盖装入帽架并打开管。将帽架固定在顶盖上, 确保管倒。确保胶盖材料突出于瓶盖的尖端, 以保证所需的组织得到适当的捕捉。</li>
	<li>在 "软件" 面板中选择 "手动" 解剖模式。仔细检查 TM (图 5B) 是否直接位于隔离管下面。设置白平衡并调整亮度以获取最佳图像质量 (图 4)。</li>
	<li>要开始切割, 手动调整焦点, 并绘制一条线使用手绘工具, 确保收集帽保持在尚未触及膜的向上位置。在 TM 满足巩膜的地方绘制部分圆圈 (图 5C), 然后按 "剪切"。一旦第一次切割完成后, 将 cap 定位到下一位置并重新调整焦点, 然后在打开或自由空间中绘制两条线, 以连接在 TM 周围完成圆圈的两个部分圆, 然后按 "剪切" 并从剖面收集组织。, 确保 TM 由显微切割帽 (图 5 d F) 拾取。</li>
	<li>将 cap 重新定位到向上位置, 然后在其余部分重复 (5.2-5.8)。稍微调整 cap 位置, 以便所有独立的示例都适合于 cap 而不重叠 (图 5G)。为了保持一致性, 一张幻灯片的时间窗口是20分钟. 如果需要更多的时间从所有部分隔离 TM, 则每只宠物幻灯片使用较少的部分。对于下一个 PET 膜, 插入一个新的隔离管并重复5节。</li>
</ol>6. Microdissected TM 组织的裂解
<ol><li>新鲜制备 RNA 裂解缓冲液, 溶解 LCM 分离组织。添加10µL β巯基乙醇到每1毫升的裂解缓冲。在室温下贮存溶解缓冲液, 巯基乙醇不超过1月。</li>
	<li>从显微解剖开始20分钟内将隔离管从帽架上取下。用眼睛可视化瓶盖表面, 以确保捕获 TM。</li>
	<li>小心地将10µL 溶解缓冲器放在收集管的盖子上, 确保溶解缓冲完全覆盖 microdissected TM。在室温下轻轻地关闭胶盖和孵育收集管10分钟。孵化后, 离心机在 800 x g 2 分钟, 并在干冰上放置裂解。贮存裂解物在-80 摄氏度后进行提纯和分析。</li>
</ol>7. RNA 分离和质量分析
<ol><li>在室温下解冻样品, 分析 RNA 质量。将所有样本从单个鼠标分离到一个 RNase 1.5 毫升离心管。 注: 例如, 对于每一个生物复制, 产生了 10-12 管与 microdissected 帽在10µL 裂解, 并所有裂解物转移到一个单一的管每一个生物复制 (100-120 µL 裂解)。</li>
	<li>加入一卷新鲜制备的70% 乙醇 (用无 RNase 水制成) 到每个裂解液溶液中。然后, 纯化总 rna 跟随 rna 隔离套件用户指南。确保从每个 RNA 样本中去除基因组 DNA。 注意: 基因组 DNA 可能干扰下游 RNA 分析。</li>
	<li>通过将每个鼠标中的样本汇集在一起并分析核糖体 rna 完整性 (图 6A), 测量从 TM 分离出来的 rna 的质量。 注意: 由于 TM 的体积小, 在 rna 质量剖面上可能无法观察到核糖体 rna 峰值 (图 6B) (在图 6AC中观察到的两个峰值介于 1000-4000 bp 之间), 用于计算 rna 完整性数 (玲)47。<ol>
<li>通过将 rna 与膜滑动上较丰富的剩余组织分离, 获得更精确的 rna 完整性测量。为了获得一个有代表性的吹打, 从膜上的眼组织中分离出 rna, 通过将10µL 裂解缓冲到一个组织切片上, 进行 rna 分离。分析 RNA 质量并计算出 (图 6C)。</li>
	</ol>
</li>
	<li>对于下游 RNA 应用, 使用低输入 rna 试剂盒进行测序和 cDNA 文库的生成, 以产生可重现的结果, 从80段的 LCM 分离 TM。</li>
</ol>8. 分析
<ol><li>为了验证所收集的组织实际上是丰富的 TM 相关基因, 收集多个额外的 RNA 从 microdissected 全眼, 巩膜, 虹膜, 视网膜, 角膜和透镜从3个单独的小鼠。使用此 RNA 结合 LCM 收集的 rna 从孤立 TM 和睫状体收集从4单独的小鼠。</li>
	<li>用 cdna 反向转录试剂盒将 RNA 从 microdissected 样品转化为 cdna。从 LCM 分离样品中放大 rna, 用低输入 RNA 转化为 cdna 试剂盒。</li>
	<li>分析所有 RNA 的小梁网表达基因, myocilin (MYOC) 和 alpha-actin-2 (ACTA2), 并规范化使用HSP90a1管家基因的数字 PCR (图 7A-B)。</li>
</ol>

作者没有什么可透露的。

TM 在积极维持稳态眼压中起着至关重要的作用, 其功能障碍被广泛接受为高血压青光眼的主要病因1,2,19。GWAS 分析发现的几个基因中的单核苷酸多态性与青光眼风险的增加和对 TM 流出设施的阻力增加有关;然而, 导致这种疾病的精确分子机制尚未完全理解21,22,23</su...

青光眼是一组以视神经病变和视网膜病变为特征的疾病, 最终导致不可逆转的失明1,2。据估计, 到 2020年, 全世界将有7000万人生活在某种形式的疾病中3,4,5,6,7。原发性开角型青光眼 (POAG) 是最普遍的青光眼类型, 其特点是水中幽默 (AH) 流出导致增加眼压 (眼压)8,9,10, 11,12,13,143,15,16,17,18。左不治疗, 长期升高的眼压导致视网膜和视神经头的渐进和不可逆转的损害导致桡盲症1,2,19。目前所有减缓青光眼进展的方法都集中在降低眼压, 或者通过降低睫状体的产率或增强其流出1,8,9,10,11,12,13,14. 小梁网 (TM) 在积极调节原发性 AH 流出通路中起着至关重要的作用, 其功能不当是高血压性青光眼1,2,19的诱因。然而, 与 TM 功能障碍相关的分子机制以及它如何调节 AH 引流尚未完全理解, 目前是青光眼研究的主要焦点1,2,19,20. 虽然一些基因组范围的联合研究 (GWAS) 已经将一些基因与青光眼联系起来, 而且对 TM 流出设施的抵抗力增加, 但导致疾病的确切分子机制还没有完全理解21,22,23,24,25。
动物模型大大增强了我们目前对青光眼疾病进展的认识 (在3,15,16,26,27,28, 29,30,31,32,33)。研究 TM34、35、36的一些开创性方法已经被广泛应用, 以促进我们目前对正常和病态组织的理解。尚未广泛探讨的一个领域是利用转基因小鼠模型研究 TM 失败的分子机制。转基因敲除和敲出小鼠研究 tm 相关基因, 如 Myocilin (Myoc)37,38和Cyp1b139, 一直是研究 tm 分子机制的主要工具。功能。可以理解的是, 小鼠 TM 的微小尺寸代表了一个严重的障碍, 必须克服, 才能开始研究这种组织。老鼠模型是研究疾病遗传学和分子机制的有力工具, 而 LCM 技术的进步提供了必要的工具, 以授权研究最小和最微妙的组织, 包括 TM。
在本报告中, 提出了一种健壮的、可重现的方法, 用于从小鼠眼中提取高浓度 TM 的 LCM, 并与随后的 RNA 分离进行比较, 并对下游表达进行放大。类似的方法已经成功地用于小鼠分离其他类型的眼睛组织40,41,42,43,44, 本文所报告的方法可以应用到其他眼睛的离散组织研究 RNA, microRNA, DNA 和蛋白质。重要的是, 这种技术可以利用转基因小鼠更好地了解 TM 损伤的分子机制在青光眼和眼部疾病中的作用3,15,16,17 ,18,26,31,45,46。通过 LCM 分离小鼠眼 TM 的能力将是进一步深入了解几种眼部疾病分子机制的有用技术。

<table><tbody><tr><td>ACTA2 ddPCR Primers (dMmuCPE5117282)</td><td>BioRad</td><td>10031252</td><td>FAM</td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td>G2946-90004</td><td></td></tr><tr><td>Agilent RNA 6000 Pico kit</td><td>Agilent Technologies</td><td>5067-1513</td><td></td></tr><tr><td>BioRad QX200 Droplet Digital PCR System</td><td>BioRad</td><td></td><td></td></tr><tr><td>Small Paint Brush</td><td></td><td></td><td></td></tr><tr><td>Charged Glass Microscope Slide</td><td>Thermo scientific</td><td>4951PLUS-001</td><td></td></tr><tr><td>Cresyl Violet Acetate</td><td>Sigma Aldrich</td><td>C5042</td><td></td></tr><tr><td>Curved Scissors</td><td></td><td></td><td></td></tr><tr><td>Eosin Y dye</td><td>Thermo scientific</td><td>71204</td><td></td></tr><tr><td>Ethanol</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td>Curved and Serrated tip (preferred tip size: 0.5 x 0.4 mm)</td></tr><tr><td>HemaCen</td><td>American MasterTech</td><td>STHEM30</td><td></td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>Applied Biosystems</td><td>4368814</td><td></td></tr><tr><td>Hsp90a ddPCR Primers(dMmuCPE5097465)</td><td>BioRad</td><td>10031255</td><td>VEX</td></tr><tr><td>Leica CM1850 Cryostat</td><td>Leica</td><td></td><td></td></tr><tr><td>Millex-GS filter unit</td><td>EMD Millipore</td><td>SLGS033SB</td><td>0.22 &amp;micro;m</td></tr><tr><td>MMI CellCut UV Cutting Model</td><td>Molecular Machines &amp;amp; Industries</td><td></td><td>LCM intrument</td></tr><tr><td>MMI CellTools Software</td><td>Molecular Machines &amp;amp; Industries</td><td>50202</td><td>LCM software</td></tr><tr><td>Sample Tube for Laser Capture Microdisssection</td><td>ASEE Products</td><td>ST-LMD-M-500</td><td>Isolation Cap Tube/Manufactured by Microdissect GmBH in Germany and distrubted by ASEE Products</td></tr><tr><td>Sample Tube for Laser Capture Microdisssection (Alternative)</td><td>Molecular Machines &amp;amp; Industries</td><td></td><td></td></tr><tr><td>modified Harris Hematoxylin</td><td>Thermo scientific</td><td>7211</td><td>FAM</td></tr><tr><td>MYOC ddPCR Primers (dMmuCPE5095712)</td><td>BioRad</td><td>10031252</td><td></td></tr><tr><td>PBS</td><td></td><td></td><td></td></tr><tr><td>Memebrane Slides, RNase Free</td><td>ASEE Products</td><td>FS-LMD-M-50r</td><td>Polyethylene terephthalate (PET) membrane/Manufactured by Microdissect GmBH in Germany and distrubted by ASEE Products</td></tr><tr><td>Memebrane Slides, RNase Free (Alternative)</td><td>Molecular Machines &amp;amp; Industries</td><td>50102</td><td></td></tr><tr><td>Rapid Fix</td><td>Thermo scientific</td><td>6764212</td><td>H&amp;amp;E staining</td></tr><tr><td>RLT Buffer</td><td>Qiagen</td><td>79216</td><td>lysis bufffer used for LCM samples</td></tr><tr><td>RNAseZap</td><td>Sigma</td><td>R2020</td><td>RNase decontamination solution</td></tr><tr><td>Protect RNA RNAse Inhibitor</td><td>Sigma Aldrich</td><td>R7397</td><td></td></tr><tr><td>RNeasy Micro Kit</td><td>Qiagen</td><td>74004</td><td>RNA isolation kit</td></tr><tr><td>SMART-Seq v4 Ultra Low Input RNA Kit</td><td>Takara Clontech</td><td>634888</td><td>low input RNA to cDNA kit for LCM samples</td></tr><tr><td>SuperMix (no dUTP)</td><td>BioRad</td><td>1863023</td><td>digital PCR master mix</td></tr><tr><td>Tissue-Tek Cryomold (25mm x 20mm x5mm)</td><td>Sakura</td><td>4557</td><td></td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>Stratalinker UV Crosslinker</td><td>Stratagene</td><td>400075</td><td></td></tr><tr><td>Xylene</td><td>Macron</td><td>8668</td><td></td></tr></tbody></table>

laser capture microdissection of highly pure trabecular meshwork from mouse eyes for gene expression analysis

激光捕获显微切割 (LCM) 允许基因表达分析的单细胞和丰富的细胞群体在组织切片。LCM 是研究细胞分化的分子机制和各种疾病, 包括青光眼的发展和进展的重要工具。青光眼, 包括一个进步的光学神经病家族, 是世界上最常见的不可逆转的失明原因。结构变化和损伤内小梁网 (TM) 可能导致增加眼压 (眼压), 这是一个主要的危险因素, 发展青光眼。然而, 所涉及的精确分子机制仍然不太清楚。进行基因表达分析的能力对于进一步深入了解这些细胞的功能及其在眼压和青光眼的发展中的作用至关重要。为了实现这一目的, 需要一种可重现的方法, 从冷冻切片中分离出高度丰富的 TM, 并提出一种用于下游基因表达分析的方法, 如 qPCR 和 RNA 序列。本文提出的方法是从小鼠眼中分离出高度纯净的 TM, 用于下游数字 PCR 和微阵列分析。此外, 这种技术可以很容易地适应, 以隔离其他高度丰富的眼睛细胞和细胞室, 已经很难孤立的小鼠眼。LCM 和 RNA 分析的结合可以有助于更全面地了解青光眼的细胞事件。

从4种不同的小鼠中提取 TM 和睫状体的 RNA, 以便能够分析基因表达, 并比较从三只小鼠的全眼、巩膜、虹膜、视网膜、角膜和晶状体中的表达。在所有收集到的组织中分析了 tm 表达基因、 MYOC48和ACTA249 , 以确认在 tm 中, 分离的 tm 样品确实高度丰富。由于在 LCM 样品中的 cDNA 数量极低, 采用了数字 PCR 技术, 经证明可重现性更少...

Watch this Scientific Journal Video about Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis at JoVE.com

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

在这里, 我们描述了一个可重现的激光捕获显微切割 (LCM) 的协议, 用于分离小梁网 (TM) 的下游 RNA 分析。分析 tm 基因表达变化的能力有助于了解 tm 相关眼部疾病的基本分子机制。

从小鼠眼中激光捕捉高纯大梁网的基因表达分析

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

laser-capture-microdissection-highly-pure-trabecular-meshwork-from

Research

JoVE Journal

Biology

9.2K 次观看.  National Institute of Environmental Health Sciences, NIH. 在这里, 我们描述了一个可重现的激光捕获显微切割 (LCM) 的协议, 用于分离小梁网 (TM) 的下游 RNA 分析。分析 tm 基因表达变化的能力有助于了解 tm 相关眼部疾病的基本分子机制。

视频: Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

科研

发育生物学

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

激光捕获显微切割哺乳动物组织

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

生物学

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

神经科学

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

激光诱导的慢性高眼压表征视觉缺陷的小鼠模型

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

医学

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

激光显微切割的从细胞的一个子集基因表达分析中的应用果蝇永光盘

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

用于小鼠角膜和康康菲瓦实时成像的自定义多光子显微镜平台

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

生物工程

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

在体内小鼠激光诱导脉络膜新生血管模型的多模态成像与分析

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 体内 成像

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

从小鼠眼中激光捕捉高纯大梁网的基因表达分析

摘要

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方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

激光捕获显微切割哺乳动物组织

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

激光诱导的慢性高眼压表征视觉缺陷的小鼠模型

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

激光显微切割的从细胞的一个子集基因表达分析中的应用果蝇永光盘

用于小鼠角膜和康康菲瓦实时成像的自定义多光子显微镜平台

在体内小鼠激光诱导脉络膜新生血管模型的多模态成像与分析

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空体内成像

从小鼠眼中激光捕捉高纯大梁网的基因表达分析

摘要

探索更多视频

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

激光捕获显微切割哺乳动物组织

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

激光诱导的慢性高眼压表征视觉缺陷的小鼠模型

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

激光显微切割的从细胞的一个子集基因表达分析中的应用果蝇永光盘

用于小鼠角膜和康康菲瓦实时成像的自定义多光子显微镜平台

在体内小鼠激光诱导脉络膜新生血管模型的多模态成像与分析

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 体内 成像

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空体内成像