We thank Dr. Vicky Macias, Angeline Giangreco and Avani Vaishnav for assistance with optimizing this methodology over the years and Yang Zhang and Dr. Jian Ma at the University of Illinois at Urbana-Champaign for the RNA seq analysis. This work was supported by NIH/NCI R01 CA166588-01 (Nonn), American Cancer Society Research Scholar 124264-RSG-13-012-01-CNE (Nonn), NIH/NCI R03 CA172827-01 (Nonn), DOD-CDMPR PRCP Health Disparities Idea Award PC121923 (Nonn) and a Prevent Cancer Foundation grant (Zhou).

通过机构审查委员会批准的协议和/或豁免在伊利诺伊大学芝加哥分校获得了用于这些实验的所有人体组织。 1.第新鲜冰冻前列腺到PEN-幻灯片，到带电玻片<ol><li>前一天或切片样前几个小时，清洁工具（即，刷，镊子，coplins，叶片，PEN框载玻片（如果不是已经无RNase），一个ETOH安全标记和铅笔）和一个内室温的低温恒温器，使用喷雾瓶和实验室湿巾RNase的去污溶液。 <ol><li>允许RNase的去污溶液坐5分钟擦去前，用depcH 2 0冲洗，以除去遗留RNase的去污溶液中，用70％乙醇的最终漂洗（制备有depcH 2 0）。 注意：重​​要的是要保持无RNase的工作区。所有coplins，工具，冻结的安装中，叶片和使用的幻灯片，应仅用于核糖核酸酶免费工作与从未使用在标准的非无RNA酶的环境。所有仪器必须与每个实验之前核糖核酸酶去污溶液进行处理。 </li></ol></li><li>低温恒温器冷却至-24℃。 </li><li>将下面的清洁无RNA酶的仪器内的低温恒温器，以至少30分钟冷却切片之前：滑动科普林填充了100％的乙醇，工具，小型冷冻组织盒和安装卡盘。 </li><li>检索cryostorage和地方冷冻前列腺组织成一个干冰填充泡沫桶运输。放置入组织冷冻切片机平衡到低温恒温器温度为至少30分钟。 注意：人类前列腺组织的激光捕获显微解剖可以是新鲜冷冻或福尔马林固定石蜡包埋（FFPE）。时间紧迫和幻灯片准备是组织源之间略有不同。在这里，我们描述的步骤进行冰冻切片。 </li><li>有一个组织在工作时间和喷射冰冻安装介质插入戈巴托使用冷冻镊子盒的米，并迅速装入冷冻的前列腺组织进入冰冻封固剂。 </li><li>放置与组织盒到冷却器中的低温恒温器5分钟以设定冷冻封固剂。 </li><li>喷射少量冰冻安装介质上的吸盘和高达小心地按上的组织下侧到冰冻的安装介质。让在低温恒温器设定为5分钟，以允许冷冻封固剂完全固化。 </li><li>将卡盘插入支架，并拧紧。 </li><li>锁定手柄锁定位置，并把新刀片到低温恒温器。为下游分子端点包括扩增步骤（即，定量PCR），建议使用一个新的刀片针对每个病人，以防止污染（或移 ​​动刀片转移到为下一个样品使用叶片的面积）。 </li><li>解锁手柄，通过用手或用脚踏小心推动组织到连和暴露组织用50μ米的部分，直到所需的组织暴露为止。 </li><li>调节低温恒温器至5μm，切一个或两个部分，并将其放置到充电载玻片用于苏木精和伊红（H＆E）染色（参见步骤2）。 </li><li>立即滑动放入冷100％乙醇2分钟。 </li><li>从乙醇中除去带电的载玻片上并让它干燥在RT前进到H＆E染色（步骤2）之前。 </li><li>将RT PEN帧幻灯片的帧支持。 </li><li>低温恒温器调整到10微米和地点节到RT PEN帧幻灯片。一到四个部分可以放置到根据组织规模每张幻灯片。 </li><li>立即沉浸滑动进入冷100％乙醇2分钟。 </li><li>从乙醇中取出笔帧幻灯片，并在幻灯片中的幻灯片存储在干燥器中在-80℃冷冻，直到准备LCM。三天的最大存储时间为最佳RNA回收。 </li><li>继续执行步骤2只收取玻璃滑即继续执行步骤3笔帧幻灯片。 </li></ol> 2. HE染色-Y（H＆E）染色的组织学标记机注：本如果在充电载玻片上组织不是LCM笔帧幻灯片。 <ol><li>通过梯度乙醇水合物浸渍幻灯片上带电载玻片的部分如下：在70％乙醇浸滑动在100％乙醇两次持续3分钟，在95％乙醇两次持续3分钟，一次3分钟和两个在蒸馏的 H 2 O时间3分钟。 <ol><li>制备酸醇溶液99毫升70％乙醇+ 1 ml的1％HCl和0.1％碳酸氢钠溶液：0.1克碳酸氢钠在100毫升蒸馏水H 2 O的</li></ol></li><li>浸在苏木吉尔II（0.4％），滑动5分钟。 </li><li>在稳定运行阶H 2 O 3分钟洗掉多余的污点。 </li><li>浸滑酸醇10倍</li><li>在自来水H 2 O的3分钟清洗的幻灯片。 </li><li>浸滑在0.1％的碳酸氢钠溶液30-60秒转动紫色颜色为蓝色。 </li><li>在自来水H 2 O的3分钟清洗的幻灯片。 </li><li>漂洗在95％乙醇的滑动用10骤降。 </li><li>浸滑的曙红-Y 2-3次，持续15秒的总。 </li><li>通过梯度乙醇脱水幻灯片如下：沾下滑95％乙醇的两倍，在100％的乙醇两次，两次二甲苯（确保它表示彻底脱水的组织出现明显的）。 </li><li>安装有非水永久硬封固剂和盖玻片的幻灯片。 </li><li>让滑动干燥30分钟。 </li><li>扫描幻灯片与任何整个幻灯片扫描仪和打印大型，高分辨率图像到8“×11”的纸张。 </li><li>感兴趣的领域纲要用记号笔（通常由一个委员会认证的病理学家完成）。此标记是LCM的程序指南。 </li></ol>笔帧幻灯片3.甲苯胺蓝染色注：钍在完成当天的LCM。使用depcH 2 O水中所有的解决方案。完成在无RNase区域的所有步骤。所有coplins必须用RNase-去污溶液进行清洗。 <ol><li> LCM的天除去从-80℃冷冻室和水合物的PEN-帧滑动通过缓倾滑入90％乙醇2分钟，然后75％乙醇2分钟。 <ol><li>制备0.5％甲苯胺蓝溶于在分子生物学级水，然后过滤灭菌，通过0.2微米的过滤器，并储存在室温。此溶液可以被重新使用长达6个月。注：强烈建议准备的溶液，作为过滤可以采取几个小时之前。 </li></ol></li><li>由缓倾滑入0.5％甲苯胺蓝5-30秒的染色前列腺组织。脱色通过洗涤所述组织滑动的2倍depcH 2 O，持续15秒，随后通过75％乙醇30秒至3分钟。注意：染色和脱色时间可以调整，以保证良好的染色。前列腺往往超过污点。 </li><l我&gt;传送PEN帧幻灯片RNA酶，无干货集装箱冰上。 </li></ol> 4.激光捕获显微解剖（LCM） <ol><li>紧接的LCM，在幻灯片在42℃温暖15分钟干滑动。干只将被处理，并在冰上其余存储直到准备为他们的幻灯片。 </li><li>打开LCM和计算机的顺序，显微镜功率，激光键，激光开关，然后计算机。启动激光显微切割软件。 </li><li>使用该软件，以控制显微镜加载幻灯片和收集管。点击“卸载样品架”，加载使用部分滑动到显微镜幻灯片持有人与组织朝下并插入在显微镜下的相应插槽幻灯片固定器，然后单击“继续”。 <ol><li>点击“卸载收集管”，标签和负载0.5 mL管到管座和在仪表相应的插槽中插入。一旦瓶盖固定，加入35微升裂解液的收集瓶盖，尽量避免气泡。然后单击“确定”。 注意：如果蒸发是一个问题，稀释缓冲如下：将25μl的裂解缓冲液与10微升depcH2O。 </li></ol></li><li>在软件中，选择幻灯片已经被放置在载片支架的位置。选择收集罩收集LCM标本。 </li><li>可视化和注重通过电脑幻灯片在10X。使用该软件通过选择“激光”，然后“校准”来校准激光。调整激光的功率，光圈和速度，选择“激光”，然后“控制”，以允许所述激光束切割完全和有效地选择的区域。重复调整，直到激光切割完全穿过组织。 </li><li>使用8“×11”病理学家加价为指导，用“画形”的工具，外接上皮的所需区域lium在视图中，并避免收集任何基质。 注：多个区域可以在一个视图中选择，但是不要点击“切形”工具移动到另一种观点认为不先切割。 <ol><li>如果一个地区没有得到完全由激光切割使用“移动和切”工具手动过目一下。继续移动物镜，以新的观点，并重复激光切割，直到滑动被完全切开或已采集的组织的所需量（通常为100良性腺体收率150-500纳克的RNA）。 （参见例如在图1A）。 </li></ol></li><li>小心地从收集架管和关闭帽，被铭记在帽裂解液和组织。短暂离心30秒，收集的液体和组织在管的底部。孵育样品在42℃进行30分钟，并储存于-80℃，直到准备用于RNA分离。 </li></ol> 5.总RNA分离与费尔之三为基础的工具包和质量分析<ol><li>解冻管在冰上，使溶液的体积为100微升。 </li><li>通过施加30微升ofLysis解滤波器的中心预润湿的RNA分离试剂盒的微过滤器，并允许它浸泡whileperforming下两步（至少5分钟）。 </li><li>加入3微升LCM添加剂溶液（试剂盒中提供），以裂解物并通过涡旋混合5秒。离心30秒在试管底部收集流体。 </li><li>离心预湿的过滤器〜30秒，最高时速去除液体。 </li><li>添加1.25体积的100％乙醇（在这种情况下，129微升）以裂解物混合物中，轻轻涡旋或吸管上下。 **此方法将恢复大和小RNA种类。 </li><li>加载样品到预润湿的微过滤器，和离心机在10,000rpm×g离心1分钟，以结合核糖核酸到过滤器。然后洗微过滤器180微升“洗涤液1，R21;离心在10,000rpm×g离心1分钟。 注：从这点上的所有离心应该13,000 XG，或最高速度来完成。 </li><li>洗涤用180微升“洗涤溶液2和3”，分别过滤两次的指示该试剂盒的协议。离心30秒，然后丢弃通过流，和自旋过滤器，持续1分钟，以除去残留的流体和以干燥过滤器。 </li><li>转移过滤到一个新的收集管。 </li><li>暖洗脱溶液至-95℃（由试剂盒提供）。 </li><li>申请10微升预热洗脱溶液在过滤器的中心，盖上并存储5分钟，室温。然后离心1分钟以洗脱RNA，并重复此步骤一次，得到〜20微升的RNA。 </li><li>进行DNA酶I处理15分钟，在37℃。 </li><li>量化的RNA通过分光光度计260nm处的吸光度。光密度在260毫微米和280毫微米波长的比率被用来评估的纯度的核糖核酸（ 见表 1）15。 </li></ol> 6.基因表达分析注意：长RNA种类（像的mRNA和lncRNAs）的基因表达被示出在步骤6.1和短RNA物质（如微RNA）示于步骤6.2。不同套件可用于RT-qPCR的和由试剂盒所需RNA的量而变化（如少至10毫微克）。 RNA测序分析在6.3节。 <ol><li>逆转录（RT）的RNA成cDNA混合5微升20毫微克/μLRNA样品与5微升RT主混合物（RT缓冲液，的dNTP的，随机引物，逆转录酶，RNA酶抑制剂）。孵育25分钟，反应在25℃，然后120分钟，在37℃，5分钟，在85℃。 </li><li>稀释RT反应1:10无RNA酶的水，并使用每10微升的qPCR反应2.5微升用引物或引物/探针组的目的基因。 注：为方便部分降解RNA分析最好是使用底漆FOř短扩增子（小于100bp）16（表2B） <ol><li>对于微小RNA表达分析运行混合5微升2-10纳克/微升RNA样品与5微升RT主结构的RT反应。 注意：每个市售基于PCR微小RNA检测技术需要使用的专有和特异性RT引物。 </li></ol></li><li>可替代地或除了RT-PCR，使用下一代测序（RNA测序或小核糖核酸-SEQ）来量化不同物种的RNA。通常情况下，使用500ng的RNA用于此过程（见表3）和实施该方法由生产和检测仪所指示的。 </li></ol>

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The authors have nothing to disclose.

基因表达的来自人的标本分析可以是具有挑战性的，不仅对质量或组织的可用量，同时也为存在于一个给定的组织标本的各种组织学实体。这是在其中良性组织基本上是间质组织和癌症领域是不含基质的前列腺特别具有挑战性。 LCM促进前列腺间质和上皮的RNA物理分离为两种不同的细胞类型（图1A）的更精确的签名。在比较macrodissection或全部组织处理，LCM可以分离细胞区室中良性组织?...

前列腺是一种异质性组织安排在腺体腺泡由肌纤维间质主要由平滑肌组成1包围分泌上皮细胞组成。上皮隔室由五个不同但有组织的细胞类型：基底细胞，分泌细胞，神经内分泌细胞，短暂扩充细胞和干细胞2。在前列腺癌（PCa），它产生于腔上皮细胞，腺癌的生长会导致基质室 3的一个明显的渐进性下降。由于这些原因，组织标本将具有在基质和上皮细胞类型的基于前列腺癌的程度的比例有明显的差异。这些差异会导致从整个组织，不考虑所期望的细胞类型的显微解剖获得的基因表达数据的偏颇的假设。因此，为了消除这种偏差，必须事先对RNA提取和分离的细胞类型基因表达分析。 Macrodissection或显微切割可用于物理地从周围基质4 -6分离充分表征的上皮区域。 Macrodissection典型的做法是与在解剖显微镜下用刀片和可以很好地用于分离大前列腺癌从基质结节，但是不能够从周围的基质中除去良性上皮的（参见实施例良性前列腺组织学于图1）。显微切割用激光（LCM）比macrodissection显著劳动密集型的，但可以非常精确地解剖良性上皮4。 从我们的实验室最近的出版物已经表明，核糖核酸可以成功地通过从任一福尔马林固定石蜡包埋（FFPE）的活检或冷冻的组织4,7-9 LCM萃取。在LCM-RNA提取的主要挑战是：1）精确解剖组织的所需区域，和2）以保持ř液晶显示模块和分离过程4,10中NA的完整性。从纯的细胞群中分离RNA可通过多种方法，包括反转录定量PCR（RT-qPCR的）7,8，微阵列11，和深-sequencing 12-14中可用于基因表达分析。 该协议的目的是从隔离LCM前列腺上皮的总RNA从冷冻的组织为下游基因表达分析。

<table>
	<tbody>
		<tr>
			<td>RNase-AWAY&#160;</td>
			<td>MBP</td>
			<td>7005-11</td>
			<td></td>
		</tr>
		<tr>
			<td>PEN-membrane 4,0 mm slides&#160;</td>
			<td>Leica</td>
			<td>11600289</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides, Superfrost Plus</td>
			<td>FisherBrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon labs.</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC (diethyl pyrocarbonate)</td>
			<td>Sigma</td>
			<td>D-5758</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplins (Staining jar)</td>
			<td>IHCWORLD</td>
			<td>M900-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplins (Staining rack)</td>
			<td>IHCWORLD</td>
			<td>M905-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Aperio ScanScope</td>
			<td>Aperio(Leica)</td>
			<td>ScanScope&#174; CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>Fluka</td>
			<td>89640-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Microdissection System</td>
			<td>Leica</td>
			<td>LMD7000</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL Thin-walled Tubes for LCM</td>
			<td>Thermo Scientific</td>
			<td>AB-0350</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAqueous&#174;-Micro Total RNA Isolation Kit</td>
			<td>Ambion</td>
			<td>AM1931</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Scientific</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 Fluorometer</td>
			<td>Life Technologies</td>
			<td>Q32866</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>Applied Biosystems</td>
			<td>4368814</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>Universal cDNA Synthesis Kit II, 8-64 rxns</td>
			<td>Exiqon</td>
			<td>203301</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan microRNA RT kit</td>
			<td>Applied Biosystems</td>
			<td>4366597</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>Hematoxylin stain&#160;</td>
			<td>Ricca Chemical Company</td>
			<td>3536-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin-Y</td>
			<td>Richard Allan Scientific&#160;</td>
			<td>7111</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser-capture microdissection of human prostatic epithelium for rna analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

在先前的研究中，我们展示了采用LCM来收集上皮细胞和间质组织由基因和微RNA的RT-qPCR的冷冻和FFPE前列腺组织来自同一患者4比较表达谱。 LCM非常耗时，尤其是当大量的RNA被收集新一代测序分析。因此，关键的是要保留的工作空间和工具无RNA酶。建议检查收集（更详细地在下面的段落中讨论）的RNA的质量和细胞特异性的控制。 图1显示之前和之后激光俘获良性上皮在显微镜下PEN...

Watch this Scientific Journal Video about Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis at JoVE.com

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

人类前列腺上皮的激光捕获显微切割的RNA分析

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

laser-capture-microdissection-human-prostatic-epithelium-for-rna

Research

JoVE Journal

Medicine

13.3K 次观看.  University of Illinois at Chicago. The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

视频: Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

人类蛋白质组分析玻璃体元素剖析

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

科研

医学

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

从手术标本的人类胰岛细胞激光显微切割的改进方法

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

人类前列腺上皮细胞形成的使用鼠类泌尿生殖窦间质细胞和人类干细胞的组织重组

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

肾小囊和异种移植皮下颗粒植入前列腺癌发生和良性前列腺增生症的评价

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

通过鱼眼研究糖尿病: 成人 tg (fli: EGFP) 斑马鱼视网膜血管的显微解剖、可视化和分析

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the quality of life. BPH includes 2 major treatments: medication and surgery. Surgical treatment is often the most effective and final intervention. Holmium laser enucleation of the prostate (HoLEP), one of the most efficient surgical procedures for BPH, is conducted transurethrally. During surgery, the most important and most difficult part is to locate the surgical capsule of the prostate, which is consistent to the idea of anatomic surgery. Different skills may be needed in treating different parts of the prostate. HoLEP tends to be efficient and safe with good hemostatic properties, and able to treat bladder calculus, which may be the complications of BPH. The technique is particularly suitable for prostate of varying volumes and sizes. Indeed, HoLEP also presents certain disadvantages, such as a long learning curve and costly equipment. Regardless, this method may be the "new standard" and the excellent method for the surgical treatment of BPH.

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate HoLEP.

Here we present a safe and efficient protocol, holmium laser enucleation of the prostate, to treat benign prostatic hyperplasia.

良性前列腺增生的外科治疗: 钬激光前列腺摘除术 (计画)。

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

激光捕获显微切割液相色谱质谱 (LCM-LC/MS) 对肺结核患者药物的空间量化

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

聚乙胺包覆氧化铁纳米粒子作为载体, 在体外和体内向巨噬细胞输送小干扰 rna

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

从人内脏脂肪组织中分离出活的脂肪细胞和基质血管组分，适用于 RNA 分析和巨噬细胞表型分析

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...