我们要感谢我们的同事提供帮助，建议和重要的投入在这个项目的各个步骤。生产视频文章从IMIDIA（ <a href="http://www.imidia.org" target="_blank">http://www.imidia.org</a> ），德国联邦教育和研究部（BMBF）德国糖尿病研究中心（， <a href="http://www.dzd-ev.de" target="_blank">http://www.dzd</a> DZD的资金支持<a href="http://www.dzd-ev.de" target="_blank">ev.de</a> ）和大学医院的卡尔·古斯塔夫·卡鲁斯在德累斯顿技术大学。导致本出版物的工作已获得创新药物计划联合执行体的支持下赠款协议N°155005（IMIDIA），资源的财政贡献，这是由欧盟的第七框架计划（FP7/2007-2013）和EFPIA公司的实物捐助。

1。冻结的人类胰腺组织<ol><li>取出脂肪组织，血管，神经和非实质组织，用手术刀和镊子，并切断胰腺组织成片（约0.5-1厘米立方体）。 </li><li>胰腺组织一块在中心，一个Cryomold，并覆盖它完全用冷水组织康OCT复合。将Cryomold成一个jar的底部覆盖有预冷却的2 - 甲基丁烷和管理单元在液氮中冷冻。在-80℃下冷冻样品存放</li></ol> 2。冰冻切片的制备<ol><li>戴上手套，在所有步骤，以避免变性的RNA。 </li><li>清洁用冷水100％的乙醇和RNase客场刀低温恒温器，样品架和画笔。将冰冻组织内的低温恒温器。 </li><li>等待，直到组织，刀，刷子达到-20°C。标签的Superfrost Plus幻灯片和预冷却，低温恒温器内室，而等待组织达到-20℃。 </li><li>应用组织康OCT化合物的试样架和将冰冻组织之上。 </li><li> ，组织康OCT复合一旦被冻结修剪cryoblock和删除任何多余的组织康OCT复合用刀片。 </li><li>剪下共连续40 10μm的切片，从胰腺组织块。此外，我们还建议要保存图纸，可以方便的识别切片的显微切割过程中的胰岛细胞内的胰腺部分概述第一和中间片切割。 </li><li>传送部从刀片的中心通过触摸的背面侧的滑动用手指（覆盖的手套箱）只升温到的区域的部分，应当坚持一个的Superfrost Plus滑动。 </li><li>干燥的部分1-2分钟内的低温恒温器在-20°C，之后，你把它们放到一个滑动箱置于干冰上。存储的部分在-80℃下</li><li>如有必要，请用纸巾和100％冷乙醇刀。 </li></ol> 3。冰冻切片脱水<ol><li>准备试剂：倒入30毫升新鲜制备的70％的乙醇，30毫升DEPC-处理水，2×30毫升100％的乙醇和30毫升二甲苯入50ml蜂星管（猎鹰）;寒意所有试剂（除二甲苯）并保持在冰上。 </li><li>用100％乙醇和RNaseZAP清洁引擎盖下的工作空间。 </li><li>过程2冷冻切片如下：固定在冰冷的70％乙醇中30秒，用5-6快速蘸冰冷DEPC-处理水，脱水两次冰冷的100％乙醇中的1分钟，在二甲苯中孵育4分钟在室温下（25℃），并空气干燥3-5分钟。重复此步骤，与另一对冰冻切片。 </li><li>将两个干冰冻切片，背靠背用锡纸包裹，以保护他们免受光照和水分到50毫升蜂星管。 </li></ol> &lt;p类= jove_title“&gt; 4。蔡司PALM微束，β-细胞的激光显微切割<ol><li>清洁的显微镜，RNaseZAP，并安装胶盖在RoboMover。 </li><li>进行以下设置：过滤器设置09（激发波长450-490 nm，发射515 nm处），AutoLPC模式，50％的激光能量，65％的激光焦点，100％的激光测速，5微米之间的距离AutoLPC拍摄，6微米的距离线，工作高度：-10,100。 </li><li>一张幻灯片插入到舞台。 </li><li>找到β细胞的自体荧光显微镜下可视（5倍或10倍镜头）。 </li><li>切换到40倍的镜头，与“写意”选择工具选择的β细胞，他们利用激光显微切割。 </li><li>捕捉四个脱水冰冻切片组织到一个AdhesiveCap。 注释1：解剖一个脱水冷冻切片的β细胞的时间不应该超过10-20分钟，以避免补液的组织切片，这将导致RNA的降解。 注释2：验证成功后，通过​​目测显微切割过程阶段的检查点。 </li></ol> 5。裂解染色体富含β细胞的组织<ol><li>删除从RoboMover AdhesiveCap。 </li><li>吸取10μL提取缓冲液（XB，PicoPure RNA提取试剂盒，大角星）到的AdhesiveCap盖下培养倒挂在42°C 30分钟。 </li><li>自旋向下在10,000 xg和把裂解干冰。将它存储在-80℃，直至进行RNA提取。 </li><li>重复步骤3）〜5）。与所有其他部分。 </li></ol> 6。 RNA提取<ol><li>将所有10个10微升裂解液</li><li>继续进行，通过在室温下工作根据协议的RNA分离试剂盒，大角PicoPure，使用以1:1的比例至70％乙醇（包括DNA酶处理）的提取缓冲液（XB）的RNA分离。 </li></ol> 7。 RNA分析<ol><li>使用1微升RNA的质量和数量，使用Agilent 2100 Bioanalyser（PicoChip公司）进行分析。定量评价是参照装在芯片上的平行的标准RNA。 </li></ol>

<ol>
	<li>Bonner, R. F., Emmert-Buck, 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+capture+microdissection:+molecular+analysis+of+tissue.">Laser capture microdissection: molecular analysis of tissue.</a> Science. 278, 1481-1483 (1997).</li><li>Suarez-Quian, C. A., Goldstein, S. R., 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+capture+microdissection+of+single+cells+from+complex+tissues.">Laser capture microdissection of single cells from complex tissues.</a> Biotechniques. 26 (2), 328-3235 (1999).</li><li>Espina, V., Wulfkuhle, J. D., 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-capture+microdissection.">Laser-capture microdissection.</a> Nature Protocols. 1, 586-603 (2006).</li><li>Mikulowska-Mennis, A., Taylor, T. B., 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>B&#246;tticher, G., Sturm, D., Ehehalt, F., Knoch, K. P., Kersting, S., Gr&#252;tzmann, R., 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=Isolation+of+Human+Islets+from+Partially+Pancreatectomized+Patients.">Isolation of Human Islets from Partially Pancreatectomized Patients.</a> J. Vis. Exp. (53), e2962 (2011).</li><li>Marselli, L., Sgroi, D. C., 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+capture+microdissection+of+human+pancreatic+beta-cells+and+RNA+preparation+for+gene+expression+profiling.">Laser capture microdissection of human pancreatic beta-cells and RNA preparation for gene expression profiling.</a> Methods in Molecular Biology. 560, 87-98 (2009).</li><li>Marselli, L., Thorne, J., 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=Gene+Expression+of+Purified+{beta}-Cell+Tissue+Obtained+from+Human+Pancreas+with+Laser+Capture+Microdissection.">Gene Expression of Purified {beta}-Cell Tissue Obtained from Human Pancreas with Laser Capture Microdissection.</a> Journal of Clinical Endocrinology &amp; Metabolism. 93, 1046-1053 (2008).</li><li>Bottino, R., Balamurugan, A. N., 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=Response+of+human+islets+to+isolation+stress+and+the+effect+of+antioxidant+treatment.">Response of human islets to isolation stress and the effect of antioxidant treatment.</a> Diabetes. 53 (10), 2559-2568 (2004).</li><li>Abdelli, S., Ansite, J., 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=Intracellular+stress+signaling+pathways+activated+during+human+islet+preparation+and+following+acute+cytokine+exposure.">Intracellular stress signaling pathways activated during human islet preparation and following acute cytokine exposure.</a> Diabetes. 53 (11), 2815-2823 (2004).</li><li>Negi, S., Jetha, 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=Analysis+of+beta-cell+gene+expression+reveals+inflammatory+signaling+and+evidence+of+dedifferentiation+following+human+islet+isolation+and+culture.">Analysis of beta-cell gene expression reveals inflammatory signaling and evidence of dedifferentiation following human islet isolation and culture.</a> PLoS One. 7 (1), e30415 (2012).</li></ol>

没有利益冲突的声明。

我们描述了一种可靠的方法，激光显微切割（LMD）的人类胰岛，胰腺手术标本。 ，一个LMD显微镜的，这个程序可以在任何研究机构进行部分胰脏，从而提高了进入人体胰岛材料从非糖尿病和2型糖尿病患者。缺乏所提供的胰腺的胰岛分离，这一点尤其重要。有利的方面，LMD通过胶原酶消化胰岛分离相比是减少外植的组织之间的时间和处理的胰岛的β细胞的富集7，以及避免苛刻的机械和酶操...

<table border="1"><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 2 - 甲基丁烷（异戊烷） </td><td> ROTH </td><td> 3927.1 </td><td></td></tr><tr><td> AdhesiveCap（不透明，500微升） </td><td> ZEISS </td><td> 415190-9201-000 </td><td></td></tr><tr><td>蜂星管（50毫升） </td><td>格赖纳生物一</td><td> 210 261 </td><td>撑裙</td></tr><tr><td>蜂星管（50毫升） </td><td>格赖纳生物一</td><td> 227.261 </td><td></td></tr><tr><td>焦碳酸二乙酯（DEPC） </td><td> SIGMA </td><td> ð5758 </td><td></td></tr><tr><td>干冰</td><td></td><td></td><td></td></tr><tr><td>无水乙醇</td><td> VWR </td><td> 20821.310 </td><td></td></tr><tr><td>液氮</td><td></td><td></td><td></td></tr><tr><td>油漆刷</td><td></td><td></td><td></td></tr><tr><td> PALM微束</td><td> ZEISS </td><td></td><td></td></tr><tr><td>剥离的方式嵌入模具（截断），12×12毫米</td><td> ProSciTech </td><td> RR12 </td><td>回内部22x22毫米，深度为21毫米</td></tr><tr><td>大角星PicoPure冷冻RNA提取试剂盒</td><td>美国应用生物系统公司</td><td> KIT 0204 </td><td></td></tr><tr><td>塑料夹</td><td></td><td></td><td></td></tr><tr><td>剃刀</td><td></td><td></td><td></td></tr><tr><td>无RNA酶的DNA酶集</td><td> Qiagen公司</td><td> 79254 </td><td></td></tr><tr><td> RNaseZAP </td><td> SIGMA </td><td> ŕ2020 </td><td></td></tr><tr><td>手术刀/手术刀片</td><td> TECHNO切</td><td> 2800111 </td><td></td></tr><tr><td>的Superfrost Plus粘连显微镜载玻片</td><td> Thermo Scientific的</td><td> J1800AMN​​Z </td><td> 25x75x1.0毫米</td></tr><tr><td>组织康OCT化合物</td><td>樱花</td><td> 4583或0807000022 </td><td></td></tr><tr><td>镊子</td><td> BRAUN </td><td> BD168R </td><td></td></tr><tr><td>二甲苯</td><td> VWR </td><td> 28975.291 </td><td></td></tr></table>

improved protocol for laser microdissection of human pancreatic islets from surgical specimens

激光显微切割（LMD）是一种技术，它允许微量的实质1,2选定的细胞和组织的恢复。解剖的细胞可以用于各种调查，如转录或蛋白质组的研究中，DNA的评估或染色体分析2,3。尤其是具有挑战性的应用LMD转录组分析，由于4 RNA的不稳定性，可以表现尤为突出，有丰富的核糖核酸酶，如胰腺组织细胞解剖时。一个显微切割技术协议，能够快速识别和收集的靶细胞，在此设置中是必不可少的，以组织处理时间缩短，因此，为确保RNA保存。 在这里，我们描述了一种协议，用于获取要用于转录研究5从手术标本的人胰岛β细胞。小件胰腺约0.5-1Çm 3的被切断从健康出现边缘切除胰腺标本，嵌入在Tissue-Tek OCT化合物中，立即冷冻冷藏2 -甲基丁烷，并保存在-80℃下，直到切片。 40连续切片厚度为10μm的低温恒温器上切下在-20°C的设置，传送单独的载玻片，在低温恒温器内干燥1-2分钟，并储存于-80℃。 紧随激光显微切割过程之前，在冰冷的固定切片，新鲜制备的70％乙醇中30秒，由冰冷DEPC-处理水的5-6蘸洗涤，并脱水，在冰冷的100％由两个一分钟的孵育乙醇后跟二甲苯（这是用于组织脱水）4分钟，组织切片，然后空气干燥，之后为3-5分钟。更重要的是，所有的步骤，但在二甲苯的潜伏期，进行使用冰冷试剂-一个修改过先前描述的协议6。 UTilization冰冷的试剂导致β细胞的内在自发荧光的显着增长，促进了他们的认可。显微切割，四个部分脱水，每次放入一个用箔纸包裹50毫升管，以保护组织的水分和漂白;，立即显微剩下的两个人。使用PALM微束仪器（蔡司）采用自动激光压力弹射模式（AutoLPC）的程序进行。的完成四个冰冻切片不再需要40-60分钟的β细胞/胰岛夹层。将细胞成一个AdhesiveCap收集，并与10μl的裂解缓冲液中裂解。每个单个RNA转录组分析用试样，得到通过组合10个细胞显微切割的样品，随后使用的Pico纯RNA分离试剂盒（大角星）RNA提取。该协议提高了内在的自发荧光的人类β细胞，从而促进他们的快速，准确识别和collection。进一步改进这个程序可以使β细胞的表型不同的解剖，更好地理解与2型糖尿病有关的变化可能产生的影响。

正如图1中所示，修改后的脱水协议导致的β细胞的自发荧光的改进相比，以前的发布的协议6。应用所描述的协议，每个39手术胰腺标本被用来生成40串行冰冻切片的平均31&#39;544&#39;704微米3组织/胰腺试样（范围：8&#39;742&#39;390 - 81&#39;522&#39;153微米3）如表1中所示。这表示大约18个胰岛的直径为150μm，或35,000β-细胞的体积。从38个不同的胰岛/胰腺试样?...

Watch this Scientific Journal Video about Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens at JoVE.com

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

激光显微切割是一种技术，使选定单元格的恢复微量的实质。在这里，我们描述了一个协议，收购手术标本用于转录组研究人类胰岛。我们的协议提高了内在的自发荧光的人类β细胞，从而有利于他们的收藏。

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 ...

improved-protocol-for-laser-microdissection-human-pancreatic-islets

Research

JoVE Journal

Medicine

14.3K 次观看.  Paul Langerhans Institute Dresden. 激光显微切割是一种技术，使选定单元格的恢复微量的实质。在这里，我们描述了一个协议，收购手术标本用于转录组研究人类胰岛。我们的协议提高了内在的自发荧光的人类β细胞，从而有利于他们的收藏。

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

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

新型抗癌药物使用手术标本肿瘤植的发展方法


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

科研

医学

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

从部分Pancreatectomized患者的人类胰岛的分离

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

人体胰腺的收集协议

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

人类胰岛染色协议

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

部分原位肝移植大鼠模型的肝动脉重建的外科手术

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl

Retinal detachment (RD) describes a separation of the neurosensory retina from the retinal pigmented epithelium (RPE). The RPE is essential for normal function of the light sensitive neurons, the photoreceptors. Detachment of the retina from the RPE creates a physical gap that is filled with extracellular fluid. RD initiates cellular and molecular adverse events that affect both the neurosensory retina and the RPE since the physiological exchange of ions and metabolites is severely perturbed. The consequence for vision is related to the duration of the detachment since a rapid reapposition of the two tissues results in the restoration of vision 1. The treatment of RD is exclusively surgical. Removal of vitreous gel (vitrectomy) is followed by the removal non essential part of the retina around the detached area to favor retinal detachment. The removed retinal specimens are res nullius (nothing) and consequently normally discarded. To recover RNA from these surgical specimens, we developed the procedure jouRNAl that allows RNA conservation during the transfer from the surgical block to the laboratory. We also standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for global gene expression analysis. The quality of the RNA was validated both by RT-PCR and microarray analysis. Analysis of the data shows a simultaneous involvement of inflammation and photoreceptor degeneration during RD.

We used retinal samples from retinectomy for a transcriptomic analysis of retinal detachment. We developed a procedure that allows RNA conservation between the surgical blocks and the laboratory. We standardized a protocol to purify RNA by cesium chloride ultracentrifugation to assure that the purified RNAs are suitable for microarray analysis.

基因表达谱分析人视网膜手术标本杂志

Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

优化高等级胶质瘤细胞培养从外科标本用于临床相关的动物模型和3D免疫化学

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

两种方法从子宫切除标本小学建立人子宫内膜基质细胞

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

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.

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 Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon dioxide and waste products. The impairment of microvascular vasomotion might be a crucial step in the development of microcirculation-related diseases. In addition, the highly vascularized pancreatic islet is adapted to support endocrine function. In this respect, it seems possible to infer that the functional status of pancreatic islet microvascular vasomotion might affect pancreatic islet function. Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion may be a feasible strategy to determine contributions that pancreatic islet microcirculation makes to related diseases, such as diabetes mellitus, pancreatitis, etc. Therefore, this protocol describes using a laser Doppler blood flow monitor to determine the functional status of pancreatic islet microvascular vasomotion, and to establish parameters (including average blood perfusion, amplitude, frequency, and relative velocity of pancreatic islet microvascular vasomotion) for evaluation of the microcirculatory functional status. In a streptozotocin-induced diabetic mouse model, we observed an impaired functional status of pancreatic islet microvascular vasomotion. In conclusion, this approach for assessing pancreatic islet microvascular vasomotion in vivo may reveal mechanisms relating to pancreatic islet diseases.

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet &#946; cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

激光多普勒: 一种测量胰岛微血管血管舒缩体内的工具

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...