我们感谢捐助者及其家人使这项工作成为可能。我们还感谢国际医学研究所和田纳西州捐助者服务机构的工作人员协助协调本研究所用组织的收集工作。这项工作得到了国家卫生研究院、nih OT2-OD023850 到 EMS2的赠款和 nih T32-DK007673 向湄京集团提供的助学金支持。我们感谢范德比尔特翻译病理学共享资源的工作人员获得 LCM 仪器和关于组织准备的建议。范德比尔特组织病理共享资源部分由 NIH 赠款 P30-CA068485-14 和 U24-DK059637-13 支持。我们感谢华盛顿大学医学院遗传学系的基因组技术访问中心, 以帮助进行基因组分析。GTAC 的部分支持的癌症中心支持补助金 #P30 CA91842 Siteman 癌症中心和信息和通信技术/CTSA 赠款 #UL1TR002345 从国家研究资源中心 (NCRR), 一个组成部分的国家卫生研究院 (nih) 和 nih医学研究的路线图。这份出版物完全是作者的责任, 不一定代表 NCRR 或 NIH 的官方观点。

此处描述的所有协议都已由范德比尔特大学机构审查委员会批准。
1. 组织到达前的准备工作
<ol><li>获得适当的 IRB 批准, 并与人体器官捐赠机构协调, 从捐赠者那里获得新切除的无固定肠道组织, 以满足研究所需的所有研究标准。 注: 本协议可适用于小鼠和其他啮齿动物模型使用。<ol>
<li>在每肠段切除后立即, 用冷冻 PBS 或组织贮存培养基彻底冲洗腔体, 以除去残留的腔内材料或粪便。</li>
		<li>在适当的生物危害容器中冲洗和丢弃废物后, 将组织浸入足够数量的组织储存培养基中 (如肠道组织体积的3-5 倍), 并贮存在单独标记的水密塑料中。容器, 浸没在冰或冷藏, 直到使用)。</li>
		<li>从收集时间起18-26 小时内处理组织, 以防止大量 RNA 降解。 注意: 根据肠道和储藏的清洁程度, 可以延长我们建议的时间限制。</li>
	</ol>
</li>
	<li>如本议定书所示, 事先准备好人体组织收集所需的所有材料 (请参阅材料表以了解详细的产品信息)。确保所有所需的个人防护设备始终佩戴。</li>
	<li>准备干冰桶连同干冰泥浆, 如图 1所示。<ol>
<li>粉碎足够的干冰, 以填补4标准圆形冰桶和一个长方形的冰桶。一个标准的桶应该有小 (11 x 21 cm) 实验室组织放置在干冰表面。如果可能, 使用新的, 未开封的实验室组织盒。</li>
		<li>在一个干净的长方形冰桶里 powderizing 2 升的干冰, 做一个干冰泥浆。</li>
		<li>添加预冷 2 MB 到干冰表面。用吸管尖端盒盖将浆料的表面混合并平整, 使浆料的形状浮出水面, 沿矩形桶的两侧由大块的干冰环绕。</li>
		<li>在冰桶的边缘放置几个薄薄的干冰块, 以鼓励更快的结冰。在一定的时间内, ≥4基模在干冰泥浆桶中应有适当的空间。<ol>
<li>可以选择, 将冰桶堆叠在另一个长方形冰桶内, 用干冰填充¼。这种定位有助于更好地隔离干冰泥浆, 并防止需要经常调整干冰和 2 MB 的过程中。</li>
		</ol>
</li>
		<li>按照以下顺序将干冰桶放置在装配线上: 1) 干冰泥浆桶, 2) 实验室组织-干冰桶, 3 & 4) 普通干冰桶, 5) 矩形干冰桶 (图 1A)。</li>
	</ol>
</li>
</ol>2. 制备闪光冷冻人体肠道组织
注: 这整个过程可以采取45-80 分钟每肠段, 取决于肠道组织质量。我们还建议收集质量控制样本, 以评估 RNA 的质量和组织学之前进行 LCM。
<ol><li>用湿冰填满一个大托盘, 在干冰上放置外科切割板。</li>
	<li>在这个设置中, 冷却一个500毫升和100毫升烧杯储存的组织和修剪的部分在冷库解决方案。在切割板上预冷的基础模具, 使他们准备接受组织。</li>
	<li>在收到新鲜的肠道组织后, 将组织输送到的培养基上倒入, 并将其保存在预冷烧杯中。在这些烧杯留出一些空间, 暂时储存肠道组织和修剪的部分, 这将在随后的步骤中准备。</li>
	<li>将肠段切开约20厘米, 提供充足的组织 (40-60 厘米2), 为下游应用和质量控制样品生成 RNA。根据组织完整性的不同, 在下游加工中完成 RNA 分离可能是可行的 (即5 厘米的肠道)。</li>
	<li>用手术剪刀从肠道中切去脂肪和结缔组织。剩余脂肪沿肠道浆膜妥协的能力, 完全扁平化组织在随后的步骤。仔细修剪组织, 避免偷脂肪和结缔组织的浆膜, 这会在结构上损害肌间丛丛, 或使组织的外部扁平化, 不均匀的切片。</li>
	<li>沿肠道标本的整个长度进行纵切口, 最好在肠系膜附件部位进行。</li>
	<li>从结肠中剥离并丢弃绦虫大肠埃希氏菌, 使其在从紧张中释放时更容易平坦。</li>
	<li>将肠道粘膜伸展到冷冻切割板上, 从组织的全长中切开1.25 厘米宽的小条。 注意: 在修剪后, 肠道组织通常会扩大, 所以这个大小应该相应地调整。</li>
	<li>暂时将小条储存在冷冻组织储存液中。 注: 我们使用贝尔泽的冷库解决方案, 因为它有一个长期的货架寿命, 是相对成本效益, 可以储存在室温, 直到需要。</li>
	<li>从冷库溶液中取出一条纸巾, 切成段 1.5-2 厘米长。</li>
	<li>迅速将肠道部分涂抹在实验室的湿巾上, 去除多余的液体, 防止在闪速冷冻过程中沿肠道浆膜形成冰晶。如果组织不够干燥, 很难从肌间丛丛中获得高质量的切片。</li>
	<li>将涂抹的肠块粘膜侧向上放置在预冷、大的一次性塑料基模上 (37 毫米 x 24 毫米 x 5 毫米)。</li>
	<li>小心地压扁每个部分, 轻轻地伸展在基模表面上的组织和使用弯曲钳轻轻按下, 并驱逐任何可能被困在浆膜下的气泡。注意, 组织扩张, 而扁平化。如果需要, 修剪线段, 并以较小的尺寸剪切其余的样品。 注:如果组织过度伸展, 这会扭曲肌间丛丛。在所有气泡被除去后, 在结冰之前评估样品的厚度。如果需要, 把标本的边缘推入内, 直到肠道样本接近其原始厚度。</li>
	<li>将所装的底座模具放置在干冰的表面上, 2 MB 的浆料。组织应在30-60 秒内完全冻结, 这取决于小肠段的大小和厚度。</li>
	<li>干燥底部的基本模具, 以消除残余 2 MB 通过快速摩擦的基础上的模具在实验室组织预冷的第二桶干冰。</li>
	<li>把干的样品转移到第三盆干冰上, 把它埋在干冰的表面, 直到它准备好包装。</li>
	<li>快速观察底部的基本模具包装前, 检查样品的质量准备。注意任何没有出现平坦或有大气泡的样品, 因为 cryosectioning 和 LCM 应该避免这样做。</li>
	<li>将样品包装在被放置在桶内的干冰上的预先标记的铝箔上, 这样在保持完全冻结的情况下, 组织发生包装。</li>
	<li>包装样品与一层塑料包装, 以减少组织脱水时, 贮存在-80 摄氏度。</li>
	<li>把样品储存在长方形的水桶里, 直到它们准备好搬到冰箱里。</li>
	<li>预标签和预冷冷藏箱在-80 °c, 以立即储存样品后收集。</li>
	<li>在进行 LCM 前, 从每个肠道部分取一小部分组织, 以评估 RNA 完整性。<ol>
<li>为每个段预先贴上2毫升无 RNase 管, 填充≥500µL 溶解缓冲液。我们建议使用 RNA 提取套件 "D", 列在材料表中。</li>
		<li>融汇一个小 (2 毫米 x 2 毫米) 肚腑在一个标准组织微均质体 (20-40 s, 直到没有组织块保持)。然后, 立即冻结在干冰上的 RNA 裂解物, 储存在-80 摄氏度, 直到进行 rna 提取。</li>
		<li>(可选) 将组织冷冻培养基 (TFM) 中的部分部分嵌入作为备份。用与本节描述的完全相同的方法制备组织切片, 但在 TFM 之前, 将样品浸入底模内。</li>
	</ol>
</li>
</ol>3. Cryosectioning
<ol><li>在 cryosectioning 之前, 准备所有必需的材料进行染色和脱水, 将所需的组织样本从-80 °c 冷藏库转移到一桶干冰中。</li>
	<li>通过设置最佳切削温度 (-18 到-22 °c) 和用100% 乙醇擦拭表面, 用 RNase 净化液处理刀片和刷子托盘, 准备恒温器。</li>
	<li>为了最好地将样品粘附到恰克, 请使用以下方法。<ol>
<li>将镊子放在干冰中至少三十年代, 然后再将肠道样品放到干燥的冰浆膜面上。</li>
		<li>将3-5 毫米的组织冷冻培养基 (TFM) 倒入一个大型恒温器标本架上, 并立即将肠道样本浆膜一侧转移到 TFM。</li>
		<li>在允许 TFM 在室温下 powderized 样品2-5 秒后, 快速将试样架转到干冰上, 然后盖上干冰。确保 TFM 仍然低于肌间丛丛的平面。 注: 理想情况下, 肠道粘膜的外表面将完全坚持 TFM 之前, TFM 开始冻结不解冻的样品。</li>
	</ol>
</li>
	<li>将标本架装入恒温器的标本头中, 将试样与恒温器刀片对齐, 这样肌间丛丛的平面将平行于切割刀片。</li>
	<li>将恒温器上的切片厚度设置为8µm。完美对准标本的目的是在每张幻灯片上获得肌间丛丛最大可能的表面积。这种厚度一般推荐为人和啮齿动物组织, 但可以调整, 根据需要。</li>
	<li>部分通过浆膜和纵肌直到到达肌间丛丛。<ol>
<li>要定位肌间丛丛, 请在幻灯片上装入一个样本部分, 并用水染料 (如1% 甲酚紫或甲苯胺蓝等) 染色切片。</li>
		<li>在40-2000X 放大的光显微镜下查看该部分, 以确定纵向和圆形肌肉层之间的连接。显微镜参数在材料表中提供。在切片开始时, 浆膜会被结缔组织的存在所标记。其余的肠系膜脂肪也可能存在沿浆膜。一张外层的纵向肌肉层然后开始。一旦到达纵向和圆形肌肉层之间的边界, 就会观察到肠道神经节的一部分。 注意: 在接下来的几节中, 肌间丛丛将变得非常明显, 在一个部分 (图 2C) 中有大量的神经节存在。如果组织在切片开始时不完全平坦, 那么在给定时间内, 每个节中只会有部分神经节出现。有时, 肠道样本可能有一个固有的不均匀的肌间丛丛, 尽管组织出现完美扁平。对于十二指肠标本尤其如此。根据我们的经验, 这些样本可能需要更长的时间来处理 LCM, 但是可以在一天的会话中收集足够的 RNA。肌间丛丛的确切位置在样品之间, 根据组织和它的准备在结冰之前可能极大地变化。</li>
	</ol>
</li>
	<li>开始收集 LCM 的串行部分, 以最佳的收集提供最大数量的神经元和胶质细胞每张幻灯片。根据试样的表面积, 可以将多个截面组合到单笔膜幻灯片上。</li>
	<li>将剖面装入笔膜幻灯片上, 在恒温器中短暂冷却5-10 秒。当安装时, 组织应只略微融化, 最大限度地保持 RNA 的完整性。</li>
</ol>4. 染色
注: 有关染色过程的概览, 请参阅表 1。所有的解决方案都是用标准的50毫升圆锥小瓶制成的。用 RNase 净化液处理管外, 似乎没有改善结果 (未显示数据)。
<ol><li>在95% 乙醇中至少提前一天准备4% 甲酚紫罗兰的饱和溶液, 并在装入注射器之前完全重新悬浮甲酚紫罗兰。 注: 如讨论部分所述, 乙醇浓度可能需要降低以有效染色。我们没有测试是否使用50% 或75% 乙醇在染色显着降低 RNA 完整性。甲酚紫罗兰是光敏的, 因而应该保护免受光。</li>
	<li>在将肌间丛丛的部分安装到幻灯片上后, 立即将该幻灯片浸入95% 乙醇 (预冷于-20 摄氏度) 的锥形瓶中, 用于三十年代。<ol>
<li>如果各节没有完全坚持上一步的幻灯片, 稍微温暖的幻灯片底部与戴手套的手 (实验室擦拭应放置在幻灯片和手套之间), 以充分遵守之前, 淹没在95% 乙醇。此解决方案可在至少3-6 张幻灯片上重复使用, 而不降低 RNA 完整性。</li>
	</ol>
</li>
	<li>将幻灯片正面放置在一个经过预处理、无 RNase 的容器8上的实验室擦拭上。</li>
	<li>预填充3毫升注射器与4% 甲酚紫罗兰和附上0.2 µm 注射器过滤器。 注:我们推荐醋酸纤维素过滤器。</li>
	<li>将6-10 滴污渍直接涂抹在组织切片8上, 孵育10-30 秒, 或在染色后保留足够的染料。染色时, 用手轻轻旋转滑动容器, 以确保组织切片均匀涂上污渍。</li>
	<li>把污渍倒掉, 然后立即在75% 乙醇瓶中把滑梯弄脏。反复淹没的幻灯片, 直到过量染料已大部分渗出的样品。这可能需要10-20 秒。</li>
	<li>在十年代, 通过反复将样品浸泡在95% 乙醇瓶中, 最终完成染色. 反复淹没样品, 直到去除所有多余的污渍。<ol>
<li>根据需要修改脱色持续时间, 以优化神经节的染色。如果去除过多的污渍, 样品变得太透明, 可能很难想象 注意: 此染色协议已根据若干出版物5、8、10、11进行了优化, 在获得满意的结果之前需要进行修改。因此, 在必要时, 应对染料中使用的乙醇浓度和脱色过程进行修改, 以获得最佳结果。</li>
	</ol>
</li>
</ol>5. 脱水
<ol><li>立即蘸100% 乙醇2-3 倍的幻灯片, 总共10-20 秒。</li>
	<li>在无水100% 乙醇中浸泡至少三十年代。由于无水乙醇是非常吸湿的, 添加分子筛 (8-12 目) 到100% 乙醇瓶之前的程序开始删除任何吸收的水, 从而更完全脱水的样本5。</li>
	<li>反复蘸二甲苯15-20 秒的滑动。此步骤完全删除幻灯片上的乙醇层。将分子筛提前添加到二甲苯管中, 充分吸附过量的乙醇和水分子5。 注意: 二甲苯被归类为眼睛和上呼吸道的刺激性物质, 应用于化学油烟机。</li>
	<li>在二甲苯中浸没滑动 (用分子筛, 8-12 目), 至少10分钟。 注:如果需要, 可以在这一步之后采取短暂的休息。样品可以留在二甲苯中4-5 小时, 不影响染色, LCM 或 RNA 的产量/完整性。</li>
	<li>(可选) 在这一点上单独准备几个额外的幻灯片。如果在所有准备好的幻灯片上完成 LCM 后准备第二轮幻灯片, 由于组织表面脱水, 恒温器中的组织可能需要 refaced。在可以收集可用节之前, 可能需要丢弃四个部分。</li>
</ol>6. 激光捕获显微切割 (LCM)
<ol><li>将滑块从二甲苯中取出, 在化学油烟机中晾干, 至少1分钟. 将 lcm 帽盒插入 lcm 显微镜阶段。将幻灯片加载到舞台上并获取幻灯片的概览图像。</li>
	<li>确定 LCM 所需的位置, 并将 cap 加载到幻灯片上。</li>
	<li>对齐红外线 (IR) 激光器并调整其功率和持续时间, 使其成为20-30 µm 直径捕获点。</li>
	<li>定位紫外线 (UV) 激光器并设置适当的切削速度和强度。</li>
	<li>概述要使用 LCM 软件收集的所需的神经节, 确保留在 cap 的可收集区域的边界内 (在软件程序的幻灯片概述中描述为绿色圆圈)。</li>
	<li>在每个跟踪中, 调整 ir 点, 以便每100-500 µm 至少有一个 ir 点。</li>
	<li>按红外线/紫外线切割按钮继续收集所有标记的神经节。</li>
	<li>一旦集合完成后, 可以将 cap 移动到新位置, 然后在质检站上重复收集过程或检查 LCM 盖, 一旦瓶盖足够填满神经节 (或直到达到60-80 分钟的建议时限)。</li>
	<li>如果盖子上有碎片, 用一支细尖的画笔擦拭它, 用 RNase 净化液预先处理, 用无核酸酶水冲洗, 然后完全干燥。</li>
	<li>在明亮的场显微镜下, 用眼睛仔细检查盖子或放大, 以确保所有碎片都被移走。如果碎片不能通过使用预处理的画笔去除, 那么使用细吸管尖 (RNase) 来刮去碎片。</li>
	<li>将 cap 固定在0.5 毫升的离心管上, 230 µL rna 裂解缓冲器 (RLT 从 rna 萃取试剂盒 "B" 中提取, β巯基乙醇加在10µL/毫升的浓度中)。</li>
	<li>反转帽, 简要涡, 室温孵育30分钟。</li>
	<li>离心机在≥ 5000 x g 为5分钟, 然后将离心管转移到干冰。 注意: 协议可以在这里暂停。rna 裂解物可以存储在-80 摄氏度, 而不影响 rna 降解至少1周。如果在同一天执行 RNA 隔离, 则在冰上冷却样品, 直到它们准备好提取。</li>
	<li>在从二甲苯中移除幻灯片后, 在建议的最大时间限制内执行所有附加集合80-90 分钟。由于脱水部分暴露于环境湿度, RNases 可以逐渐重新激活。限制收集期间可以最大限度地减少此类影响, 并最大限度地保留 RNA 完整性。</li>
</ol>7. RNA 分离
<ol><li>准备一个无 RNase 的 RNA 提取工作站。</li>
	<li>根据手册中的 RNA 提取试剂盒, 准备所有的管子和试剂。 注: 尽管 LCM 中有许多用于 rna 隔离的套件, 但我们使用 "材料" 列表中列出的 rna 提取套件 "B" 取得了最佳的成功。有关 RNA 萃取试剂的并排比较, 请参见图 5 。</li>
	<li>从-80 °c 冰箱中取出样品, 在37摄氏度的水浴中迅速解冻。</li>
	<li>立即涡解冻样品 2-3 s 均匀地分布胍硫氰酸盐。</li>
	<li>将每个样品与同等体积的70% 乙醇相结合。池2或更多裂解物在一起, 如果需要, 达到足够的 RNA 浓度。</li>
	<li>按照制造商在手册中为 RNA 提取过程的说明进行操作, 使用为 microdissected 示例优化的协议。</li>
	<li>洗脱 RNA 在最后步入最小的推荐的容量核酸酶水 (14 µL)。</li>
	<li>在 rna 分离之后, 将每个样品中的 rna 整除1-2 µL 到一个新的预先标记的无 RNase 管, 进行质量评估/质量控制, 用微流体装置, 可以想象少量的 rna, 如 Bioanalyzer。整除一个额外的1µL 进入一个单独的管量化与高灵敏度的 RNA 量化试剂盒。<ol>
<li>根据需要调整这些步骤, 以获得足够数量的 rna, 用于下游分析 (i., 在 rna 提取之前汇集更多的 LCM 盖帽)。</li>
	</ol>
</li>
	<li>将 RNA 储存在-80 摄氏度, 直到收集足够的样品进行下游分析。</li>
</ol>

<ol>
	<li>Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The second brain : the scientific basis of gut instinct and a groundbreaking new understanding of nervous disorders of the stomach and intestine. , (1998).</li><li>Grundmann, D., Klotz, M., Rabe, H., Glanemann, M., Schafer, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+high-purity+myenteric+plexus+from+adult+human+and+mouse+gastrointestinal+tract.">Isolation of high-purity myenteric plexus from adult human and mouse gastrointestinal tract.</a> Scientific Reports. 5, 9226 (2015).</li><li>Huang, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin-induced+proteome+alteration+during+cell+subculture+in+mammalian+cells.">Trypsin-induced proteome alteration during cell subculture in mammalian cells.</a> Journal of Biomedical Science. 17, 36 (2010).</li><li>Bottner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microdissection+as+a+new+tool+to+investigate+site-specific+gene+expression+in+enteric+ganglia+of+the+human+intestine.">Laser microdissection as a new tool to investigate site-specific gene expression in enteric ganglia of the human intestine.</a> Neurogastroenterology and Motility. 22 (2), 168-172 (2010).</li><li>Clement-Ziza, M., Munnich, A., Lyonnet, S., Jaubert, F., Besmond, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+of+RNA+during+laser+capture+microdissection+by+performing+experiments+under+argon+atmosphere+or+using+ethanol+as+a+solvent+in+staining+solutions.">Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions.</a> RNA. 14 (12), 2698-2704 (2008).</li><li>Hetz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+gene+expression+analysis+in+enteric+ganglia+of+human+colon+after+laser+microdissection.">Age-related gene expression analysis in enteric ganglia of human colon after laser microdissection.</a> Front Aging Neurosci. 6, 276 (2014).</li><li>. PALM Protocols - RNA handling Available from: <a target="_blank" href="https://hcbi.fas.harvard.edu/files/zeiss_labprotocol_rna_0811.pdf">https://hcbi.fas.harvard.edu/files/zeiss_labprotocol_rna_0811.pdf</a> (2011)</li><li>. Workflows &amp; Protocols: How to Use a Leica Laser Microdissection System and Qiagen Kits for Successful RNA Analysis Available from: <a target="_blank" href="https://www.leica-microsystems.com/science-lab/laser-microdissection/workflows-protocols-how-to-use-a-leica-laser-microdissection-system-and-qiagen-kits-for-successful-rna-analysis/">https://www.leica-microsystems.com/science-lab/laser-microdissection/workflows-protocols-how-to-use-a-leica-laser-microdissection-system-and-qiagen-kits-for-successful-rna-analysis/</a> (2015)</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arcturus+HistoGene+Frozen+Section+Staining+Kit:+User+Guide.">Arcturus HistoGene Frozen Section Staining Kit: User Guide.</a> Biosystems, A. , (2010).</li><li>Cummings, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+RNA+integrity-preserving+staining+protocol+for+laser+capture+microdissection+of+endometrial+cancer+tissue.">A robust RNA integrity-preserving staining protocol for laser capture microdissection of endometrial cancer tissue.</a> Analytical Biochemistry. 416 (1), 123-125 (2011).</li><li>Grover, P. K., Cummins, A. G., Price, T. J., Roberts-Thomson, I. C., Hardingham, 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=A+simple,+cost-effective+and+flexible+method+for+processing+of+snap-frozen+tissue+to+prepare+large+amounts+of+intact+RNA+using+laser+microdissection.">A simple, cost-effective and flexible method for processing of snap-frozen tissue to prepare large amounts of intact RNA using laser microdissection.</a> Biochimie. 94 (12), 2491-2497 (2012).</li><li>Heumuller-Klug, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+intestinal+mRNA:+a+matter+of+treatment.">Degradation of intestinal mRNA: a matter of treatment.</a> World Journal of Gastroenterolology. 21 (12), 3499-3508 (2015).</li><li>Gallego Romero, I., Pai, A. A., Tung, J., Gilad, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq:+impact+of+RNA+degradation+on+transcript+quantification.">RNA-seq: impact of RNA degradation on transcript quantification.</a> BMC Biology. 12, 42 (2014).</li><li>Sigurgeirsson, B., Emanuelsson, O., Lundeberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequencing+degraded+RNA+addressed+by+3'+tag+counting.">Sequencing degraded RNA addressed by 3' tag counting.</a> PLoS One. 9 (3), 91851 (2014).</li><li>Potter, S. S., Brunskill, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture.">Laser capture.</a> Methods in Molecular Biology. 886, 211-221 (2012).</li><li>Funke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microdissection+of+intestinal+epithelial+cells+and+downstream+analysis.">Laser microdissection of intestinal epithelial cells and downstream analysis.</a> Methods in Molecular Biology. 755, 189-196 (2011).</li><li>Kolijn, K., van Leenders, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+RNA+extraction+kits+and+histological+stains+for+laser+capture+microdissected+prostate+tissue.">Comparison of RNA extraction kits and histological stains for laser capture microdissected prostate tissue.</a> BMC Res Notes. 9, 17 (2016).</li><li>Muyal, J. P., Muyal, V., Kaistha, B. P., Seifart, C., Fehrenbach, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+RNA+extraction+techniques+from+frozen+and+fresh+lung+tissues:+checkpoint+towards+gene+expression+studies.">Systematic comparison of RNA extraction techniques from frozen and fresh lung tissues: checkpoint towards gene expression studies.</a> Diagnostic Pathology. 4, 9 (2009).</li><li>Mikulowska-Mennis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-quality+RNA+from+cells+isolated+by+laser+capture+microdissection.">High-quality RNA from cells isolated by laser capture microdissection.</a> Biotechniques. 33 (1), 176-179 (2002).</li><li>Pietersen, C. Y., Lim, M. P., Woo, T. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+high+quality+RNA+from+single+cell+populations+in+human+postmortem+brain+tissue.">Obtaining high quality RNA from single cell populations in human postmortem brain tissue.</a> Journal of Visualized Experiments. (30), (2009).</li><li>Guo, M., Wang, H., Potter, S. S., Whitsett, J. A., Xu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SINCERA:+A+Pipeline+for+Single-Cell+RNA-Seq+Profiling+Analysis.">SINCERA: A Pipeline for Single-Cell RNA-Seq Profiling Analysis.</a> PLoS Computational Biololgy. 11 (11), 1004575 (2015).</li><li>Adam, M., Potter, A. S., Potter, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychrophilic+proteases+dramatically+reduce+single-cell+RNA-seq+artifacts:+a+molecular+atlas+of+kidney+development.">Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development.</a> Development. 144 (19), 3625-3632 (2017).</li></ol>

作者没有披露的利益冲突。

这个过程使许多肠道神经节的有效收集成为 rna 的来源在这里, 我们加速了现有协议中概述的过程, 同时最大限度地保留了 RNA 完整性。由于这一程序中的所有步骤都是相互依存的, 因此必须在研究开始时消除所有的问题, 并尽可能以同样的方式编写所有的样本, 以获得可靠的 RNA--
从程序的开始, 如果时间间隔 (冷缺血时间) 在器官捐赠时组织的收集和接收组织在实验室中的处理时...

本方法旨在利用激光捕获显微切割技术从人体肠道组织获得高质量的肠道神经节 RNA 样品。此处描述的协议已经过优化, 以提供足够的 rna 质量和 rna 排序 (rna 序列) 的产量, 并打算用于与新鲜切除, 无固定, 闪光冷冻人类肠道组织。
功能性胃肠道和肠道运动障碍影响美国每四人之一。肠神经系统, 也称为第二脑1, 往往是这些疾病的中心, 因为它在肠道动态平衡和运动中起着关键的作用。对肠道运动的操纵一般限于手术切除 aganglionic/noncontractile 组织, 慢性饮食修饰和/或药物。令人惊讶的是, 成人的完整转录仍然是排序, 大大限制了我们的能力, 以鉴定在体内的分子, 可以被靶向药用或用于干细胞治疗。
从人肠道神经节中分离 RNA 的方法相对较少。第一种方法, 细胞离解2, 需要高的孵化温度和长时间的潜伏期;这两种都是已知的促进 RNA 降解和改变转录2,3。另一种方法, LCM, 更可靠地保存转录和保护 RNA 完整性。虽然一些研究已经使用 LCM 收集神经节从新鲜冷冻人类肠道组织4,5,6, 这些方法要么是由于 RNA 质量和数量低下, 是相当劳动密集型的, 或需要修改染色或 RNA 提取技术, 在我们的手工作。为保存在 lcm 产品手册中发现的 RNA 而设计的其他 lcm 协议提供了额外的改进7、8, 但在应用于肠神经节8的隔离时需要适应,9. 出于这些原因, 我们开发了一种基于这些资源的优化协议, 它从人类肠道神经节产生大量的高完整性 RNA, 具有相对较快的工作流, 并在大型样本数。
在本研究中, 我们提出了一个优化程序的概要, 以促进分离高完整性的 RNA 从肠道神经节从切除人肠道组织。我们的方法包含五重要方面。首先, 新切除的, 不固定的人体肠道样本应该修剪到大小, 所有多余的水分去除与实验室组织和扁平化在一个大的基础模具之前, 在干冰的泥浆和 2-丁烷 (2 MB) 的闪光冻结。其次, 肠道组织学切片应准备获得肌间丛丛在一张幻灯片上的完整平面, 它提供了一个大的肠道神经节的负载。这个步骤的成功在很大程度上取决于组织的准备过程。第三, 存在的神经节的不均匀结构要求使用聚乙烯 napthalate (钢笔) 膜幻灯片6, 这在 LCM 过程中提供了最大的速度和精度。第四、乙醇相容染料, 如甲酚紫, 应用于保存 RNA 完整性, 同时染色肠神经节。最后, rna 提取过程对于一个成功的 rna 序列的结果是至关重要的。我们寻求一种 rna 提取方法, 产生高 rna 完整性, 最大限度地提高 rna 的产量, 当开始与小收集肠道神经节, 消除 DNA 污染, 并保留尽可能多的 rna 物种。
同时, 本研究对这些因素的优化, 大大加快了肠道神经节的工作流程和产量, 具有异常的 RNA 数量和质量。结果在相当大的样本群中大致一致, 表明了这种方法的一致性。此外, 我们已经使用这些方法成功地序列了许多来自肠道神经节的 RNA 样本。这里所强调的策略也可以广泛地适应于对周围和中枢神经系统所期望的神经节或细胞核进行 LCM, 以及其他需要隔离高质量 RNA 的情况。

<table border="0" cellpadding="0" cellspacing="0" style="width:920px;" width="919">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:315px;">10% Neutral-buffered formalin</td>
			<td style="width:280px;">Sigma</td>
			<td style="width:131px;">HT501128-4L</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-Methylbutane</td>
			<td>Fisher</td>
			<td>O3551-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3-mL syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50-mL conical tubes, polypropylene&#160;</td>
			<td>Corning</td>
			<td>05-526B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Base molds 37x24x5mm, disposable</td>
			<td>Electron Microscopy Sciences</td>
			<td>5025511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Belzer UW&#160; Cold Storage Solution</td>
			<td>Bridge to Life</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CapSure Macro LCM caps</td>
			<td>Arcturus, ThermoFisher</td>
			<td>LCM0211</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cling Wrap, Press&#8217;n Seal&#160;&#160;</td>
			<td>&#160;Glad&#160;&#160;</td>
			<td>N.A.&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cresyl Violet Acetate</td>
			<td>Acros Organics</td>
			<td>AC405760025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cutting Board</td>
			<td>Electron Microscopy Sciences</td>
			<td>63308</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, 190-proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000190</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, 200-proof</td>
			<td>Pharmco-AAPER</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Microscope slides</td>
			<td>Fisher</td>
			<td>12-550-343</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gloves, extended cuff</td>
			<td>Microflex&#160;</td>
			<td>19010144</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gowns, surgical-disposable</td>
			<td>Kimberly-Clark&#160;</td>
			<td>19-088-2116</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tray, 20 L (large polypropylene sterilizing pan)</td>
			<td>Nalgene&#160;&#160;</td>
			<td>1335920B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kimwipes (large)</td>
			<td>Kimberly-Clark&#160;</td>
			<td>34120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kimwipes (small)</td>
			<td>Kimberly-Clark&#160;</td>
			<td>34133</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laser Capture Microdissection System (ArcturusXT)</td>
			<td>ThermoFisher</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leica Cryostat Chuck, large,&#160; 40 mm&#160;</td>
			<td>Southeast Pathology Instrument Service</td>
			<td>N.A.&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Light Microscope</td>
			<td>Olympus</td>
			<td>CX43</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope objective (20X)</td>
			<td>Olympus UPlanFl 20X/0.50, &#8734;/0.17</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope objective (10X)</td>
			<td>Olympus UPlanFl 10X/0.25, &#8734;/-</td>
			<td>N.A.&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Molecular Sieves</td>
			<td>Acros Organics</td>
			<td>AC197255000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclease-free water</td>
			<td>Ambion</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PicoPure RNA extraction kit</td>
			<td>Applied Biosystems</td>
			<td>12204-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pellet Pestle</td>
			<td>Kimble Kontes</td>
			<td>4621973</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PEN membrane LCM slides</td>
			<td>Arcturus, ThermoFisher</td>
			<td>LCM022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase-free tubes, 1.5 mL</td>
			<td>Ambion</td>
			<td>AM12400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNaseZAP&#160;</td>
			<td>Sigma</td>
			<td>Sigma-R2020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Kit &#34;A&#34; (PicoPure)</td>
			<td>Applied Biosystems</td>
			<td>12204-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Kit &#34;B&#34; (RNeasy&#160; Micro kit)</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Method &#34;C&#34; (TRIzol)</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA Extraction Kit &#34;D&#34; (RNeasy PLUS Mini kit)</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Splash Shield, disposable faceshield</td>
			<td>Fisher</td>
			<td>17-310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors, Surgical, 14.5 cm &#34;sharp-sharp&#34;</td>
			<td>Fine Science Tools</td>
			<td>14002-14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe filter, cellulose acetate (0.2 &#956;m)</td>
			<td>Nalgene&#160;&#160;</td>
			<td>190-2520</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Freezing Medium</td>
			<td>General Data Healthcare</td>
			<td>TFM-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylenes</td>
			<td>Fisher</td>
			<td>X3P1GAL</td>
			<td></td>
		</tr>
	</tbody>
</table>

optimization of laser-capture microdissection for the isolation of enteric ganglia from fresh-frozen human tissue

该方法的目的是通过激光捕获显微切割 (LCM) 从未固定的、新鲜切除的人肠道组织采集的肠神经节获得高完整性的 RNA 样本。我们已经确定了工作流程中的五个步骤, 对于从肠神经节获取 rna 分离的关键, 它的质量和数量足以为 rna--首先, 在准备肠道组织时, 每个样品必须有所有多余的液体去除的印迹之前, 使浆膜尽可能多地在底部的大基模具。然后在干冰和2丁烷的泥浆上迅速冻结样品。第二, 当切片的组织, 重要的是放置 cryomolds, 使肠道部分平行的全平面的肌间丛丛, 从而产生最大的肠道神经节每张幻灯片的面积。第三, 在 LCM 过程中, 聚乙烯 napthalate (笔) 膜幻灯片在收集肠神经节时, 可以提供最大的速度和灵活性来勾勒出肠道神经节的非均匀形状。第四, 对于不同的肠道神经节可视化, 与乙醇相容的染料, 如甲酚紫罗兰, 提供优良的保存相对于水染料的 RNA 完整性。最后, 为了从捕获的神经节中提取 rna, 我们观察了商业 rna 提取试剂盒中产生的 rna 数量和质量的差异, 同时消除了 DNA 污染。这些因素在当前协议中的优化极大地加速了工作流, 产生了异常的 RNA 质量和数量的肠神经节样本。

我们已经对现有的协议进行了几项改进, 通过 LCM 能够相对快速地从人体肠道样本收集肠道神经节, 达到 RNA 序列的标准。首先, 我们优化了在 2 MB (图 1B) 放置在干冰泥浆表面的大型基模中肠道组织段的快速冻结。最佳的成功在 cryosectioning 和随后 LCM 是通过放置肠段平在一个基地模子, 面对黏膜边上升 (图 1 b-1 c) 获得。确保基础...

Watch this Scientific Journal Video about Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue at JoVE.com

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化

该协议的目的是获得高完整性的 RNA 样本从无固定的, 新鲜切除的人肠道组织, 利用激光捕获显微切割 (LCM) 分离。该协议包括制备人体肠道组织、cryosectioning、乙醇染色和脱水、LCM 和 RNA 提取的闪光冷冻样品。

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

optimization-laser-capture-microdissection-for-isolation-enteric

Research

JoVE Journal

Neuroscience

8.6K 次观看.  Vanderbilt University Medical Center. 该协议的目的是获得高完整性的 RNA 样本从无固定的, 新鲜切除的人肠道组织, 利用激光捕获显微切割 (LCM) 分离。该协议包括制备人体肠道组织、cryosectioning、乙醇染色和脱水、LCM 和 RNA 提取的闪光冷冻样品。

视频: Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

染色质免疫沉淀从以下轴索损伤的背根神经节组织

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

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 ...

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 Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

神经元的分化的人类Neuroprogenitor细胞培养激光捕获显微切割

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

通过激光捕获微分路，从小鼠脊髓运动神经细胞体中生成RNA进行转录分析

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

从开发小脑显微切割分离的不同的细胞群

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

立体定向激光捕获创伤性脑区显微解剖

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

新鲜人脑组织中毛细血管的分离

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...