作者感谢捐助者的家庭和组织的专家协助参与这项研究和刘慧卿蒙哥马利罗伯特Pietras，安富，Mitali阿加瓦尔，罗山阿加瓦尔器官采购。这项工作是由青少年糖尿病研究基金会（MC-T.）对糖尿病患者的胰腺器官捐赠者的网络支持。

1。为不染节的切片<ol><li>成立水浴和标准的石蜡切片切片。正电的幻灯片和前标签使用数量的情况下，胰腺区域（样品）和幻灯片编号。广场左侧的磁带标签的切片机夹头的石蜡块。 </li><li>按照正常切片程序，节成块，直到组织均匀遇到。产生一个连续切片（4μm厚3-6（见案例提交表格， 附录1）根据初期所需的污渍）剪彩。色带单独的章节，以便拿起，和幻灯片的地方用铅笔编号顺序。表示，如果一节是由确切顺序编号部分闲置。保持相同的方向，面向左侧的幻灯片标签的录像带中发现，在幻灯片上。干燥，在室温下过夜，然后进行染色或分配给调查人员。 </li><li> RESEAL表面一层薄薄的石蜡组织和归档保存在室温或-20°C的石蜡块</li><li>对于后续的切片，连续切片保持在上述的每个级别编号。获得1个额外的幻灯片和染色H＆E的幻灯片，每块内每个〜100μM的水平。 </li></ol> 2。 H＆E染色<ol><li>然后在第一个托盘上Autostainer自动将第一个串行从每块切片幻灯片在幻灯片机架。 </li><li>选择标准的H＆E染色方案，并像往常一样进行。 </li><li>当完成染色，Autostainer自动删除和地方在引擎盖盖片，幻灯片。 </li><li>盖玻片使用的标准技术。彻底干燥和标签印刷标签（见第12条）。 </li></ol> 3。免疫组化设置<ol><li>选择双污渍和幻灯片输入号码Dako公司计划。准备的TBST和柠檬酸缓冲，抗体和其他REAG根据已废除指定的卷（ 见表1）。装入试剂托盘和幻灯片。根据制造商的建议，清洁和废物线旋转编程。 </li><li>如果手动执行这些实验，准备估计量。在湿盒中孵育幻灯片，以避免任何一步干燥的幻灯片。 Autostainer自动时，每个试剂按照相同的程序。 </li></ol> 4。免疫组化Deparaffinization和抗原检索<ol><li>在二甲苯5分钟的幻灯片。再重复一次。不要让幻灯片干任何后​​续点，直到表示，因为这会导致过度的背景和贫困染色。 </li><li>将幻灯片转让100％的乙醇为2分钟。再重复一次。 </li><li>把新鲜配制的3％的H 2 O 2的甲醇在10分钟的幻灯片。 </li><li>在90％的乙醇，然后浸幻灯片放置在90％乙醇为3分钟。 </li><li>转让滑动到70％的乙醇为1分钟。 </li><li>在去离子水冲洗幻灯片。 </li><li>在蒸笼5分钟预热蒸锅和柠檬酸缓冲。 </li><li>添加幻灯片柠檬酸蒸锅中蒸30分钟的缓冲。 </li><li>立即转移幻灯片水，直到滑入冷却至室温（约20分钟）。 </li><li>根据染色序列（ 图1）Dako公司Autostainer自动衣架转让幻灯片和运行双重染色程序。列出Autostainer自动程序的主要步骤，以便手动运行可重复。 </li></ol> 5。第一（Ki-67的CD3，胰多肽）抗体<ol><li>阻止狙击手的解决方案为15分钟的幻灯片。用TBST洗两次。 </li><li>初级抗体孵育30分钟的幻灯片。用TBST洗两次。 </li><li> MACH2羊抗鼠（Ki-67的）或抗兔（CD3 +，胰多肽）辣根过氧化物酶（HRP）聚合物孵育30中号inutes。用TBST洗两次。 </li><li> 3,3 - 二氨基tetrahydrochloride（民建联），4分钟的幻灯片。用清水洗两次。 </li><li>胰多肽孵育的幻灯片举行时，同时测定Dako公司和TBST所有后续步骤使用，而其余的幻灯片与第二组的主要抗体孵育。另外，手动检测，进行到第6条。 </li></ol> 6。第二次（胰岛素，胰高血糖素）抗体<ol><li> Deeb医师幻灯片孵育20分钟。用TBST洗两次。 </li><li>座与10％，20％的抗生物素蛋白受体阻滞剂在TBST（胰岛素）或狙击手在TBST（胰高血糖素）的正常山羊血清20分钟。用TBST洗两次。 </li><li>孵育15分钟的胰岛素或胰高血糖素抗体。用TBST洗两次。 </li><li>胰岛素：孵育30分钟，在1:300稀释生物素标记的羊抗豚鼠猪的幻灯片。用TBST洗两次。与标准VE孵育构造函数ABC - AP试剂为30分钟。用TBST洗两次。 </li><li>胰高血糖素：孵育缓冲液中的幻灯片，在30分钟，30分钟羊抗鼠碱性磷酸酶（AP）聚合物。用TBST洗两次。 </li><li>虽然幻灯片标记培养，温暖的的LPR缓冲区室温。加入1滴液体永久红（LPR），每3毫升缓冲立即使用前和Dako公司试剂架的地方。在LPC孵育4分钟的幻灯片。用清水洗两次。 </li><li>孵育1分钟的在苏木幻灯片。用清水冲洗两次。 </li><li>在TBS的pH值7.6孵育1分钟的幻灯片。用清水冲洗两次。 </li><li> DAKO公司Autostainer自动卸载的幻灯片。 </li><li>立即脱水胰多肽染色的幻灯片。对于所有的双彩色幻灯片，至少1小时，然后干燥脱水。 </li></ol> 7。脱水<ol><li>地方滑出80％乙醇1分钟。 </li><li>浸在95％乙醇的幻灯片。 </l我&gt; <li>保持在95％乙醇，30秒的幻灯片。 </li><li>幻灯片转移到100％的乙醇，并保持1分钟。重复。 </li><li>二甲苯浸幻灯片。 </li><li>把握在1分钟二甲苯幻灯片。重复。 </li><li>立即安装幻灯片使用cytoseal的盖玻片。 </li></ol> 8。幻灯片标签<ol><li>输入的情况下识别信息，包括机关和块数，染色，日期和打印。串行节号是可选的第一张幻灯片，每块污渍。随后水平表示编号。 </li><li>放置在幻灯片上的标签。 </li></ol> 9。幻灯片扫描<ol><li>放置在扫描仪的托盘彩色幻灯片，根据仪器和扫描。 </li><li>组织由捐助国和组织类型（胰腺头，身，尾，脾）的幻灯片。 </li><li>在-20°C在室温和任何剩余的未染色幻灯片存档的彩色幻灯片。 </li></ol> 10。 representative结果这些染色程序开发与糖尿病（nPOD）胰腺器官捐赠者提供基线表征石蜡和鲜冻块JDRF网络。每个捐赠者有一个基线特征进行染色从每个胰腺地区的2块（头，身，尾）和脾脏（ 图1中 ，也看到案件提交表格， 附录1）组成。 H＆E染色为临床与临床级的解决方案，用于整个工厂编程Autostainer自动进行。作为额外的捐助块切片，每个人都有为H＆E公司的幻灯片，以显示组织形态。当块切片再次，分配给调查，更深的层次也将有代表性的H＆E幻灯片记录了整个块的组织形态以及编号在每个级别的串行部分幻灯片。彩色幻灯片标记鉴定的情况下，胰腺区域，块数，染色和日期（ 图2）和扫描到网上的病理信息管理系统。从控制捐助国的代表图像显示在低和高放大倍率显示预期的结果，此过程如图3。胰多肽（D，H）的幻灯片染色检测，检测手动执行，并显示减少染色苏木染液强度在不同的日子。初级抗体染色强度或检测染液之间的差异是要避免，特别是如果使用图像分析，否则滑与需要个人色彩校正，以达到同样的细胞领域或计数。 每个实验室应优化很多，很多的变化和其他试剂和条件可能会影响染色强度和特异性抗体的浓度。随着新的收购原因男装，染色程序验证与已知阳性和阴性对照样本，以实现前试剂染色强度相同程度。在nPOD病理核心优化的额外抗体提供作为参考表2中，并已用于免疫荧光共聚焦显微镜检测multilabeling。 <img src="/files/ftp_upload/4068/4068fig1.jpg" alt="图1"/> 图1。 DAKO染色电网。本图描绘了一个从nPOD捐助胰腺上Dako公司Autostainer自动执行常规分析。 nPOD捐助者编码与一个4位数的识别号码。两个滑动托盘显示染色组织的幻灯片。替代幻灯片配置预计取决于捐助幻灯片的数字。阳性对照包括列入已知两个双污渍和捐助者的Ki-67的和CD3脾的积极捐助。阴性对照是双重的，包括两个SLIDES从一个捐助国的胰腺块从捐助者对胰岛素和胰高血糖素脾的主要抗体和两个幻灯片的宿主物种的免疫球蛋白（Ig）的培养。 <img src="/files/ftp_upload/4068/4068fig2.jpg" alt="图2"/> 图2。前进行数字扫描， 代表双彩色幻灯片。一个典型的成品幻灯片显示幻灯片标签参数和组织安排。 <img src="/files/ftp_upload/4068/4068fig3.jpg" alt="图3"/> 图3。代表的H＆E和免疫组化在胰腺癌中的部分污渍。成年女性器官捐赠者的胰腺（6096-04云台）的部分染色和数字扫描在协议执行。图像得到使用的ImageScope观看整个节（AD）和一个小岛（EH）的方案。预计胰岛胰岛素，胰高血糖素和胰多肽，染色强度D节描绘观察部分屋宇署胰头块包含所有胰岛包含主要胰多肽阳性细胞的钩突地区或腹胰叶一小部分。请注意，面板D苏木染液是太轻，而苏木染液强度B和C是最佳的。面板高血压表明，与预期的β细胞和α细胞分布背叶胰岛。一些胰多肽细胞被发现在较低的胰岛（高）的左半部分。 ，E-H &amp;E; B，F - Ki67的胰岛素，C，G - CD3 +胰高血糖素，D，H - 胰多肽。 AD，0.2X的EH-12.8x。

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没有利益冲突的声明。

免疫组化标准化程序是图像分析的关键，特别是当使用大量的幻灯片之间随着时间的推移以计算机为基础的算法。在本报告所述的免疫组化染色过程将使一批在8小时工作制Autostainer自动给定的捐赠者的样品的分析和修改是从以 ​​前的报告5。每个彩色幻灯片的全数字化的幻灯片图像可通过基于web的在线病理系统nPOD附属调查。研究人员可以审查与幻灯片一起捐助者的人口统计资料，实验?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 30％的过氧化氢 ​​（H 2 O 2） </td><td> FisherScientific </td><td> H325-500 </td><td>内源性过氧化物酶阻止</td></tr><tr><td> ABC - 碱性磷酸酶（AP） </td><td>向量</td><td> AK-5000 </td><td> AP的共轭</td></tr><tr><td>抗体稀释液</td><td> Invitrogen公司</td><td> 003218 </td><td>初级抗体</td></tr><tr><td>抗生物素蛋白受体阻滞剂</td><td>向量</td><td> SP-2001 </td><td>阻止内源性生物素</td></tr><tr><td>生物素标记的羊抗豚鼠</td><td>向量</td><td> BA-7000 </td><td>二级抗体</td></tr><tr><td>烧蓝试剂</td><td> FisherScientific </td><td> 7301 </td><td>徕卡autostaineŕ </td></tr><tr><td>柠檬酸缓冲液，pH值6.0 </td><td> BioGenex </td><td> HK086-9K </td><td>抗原检索</td></tr><tr><td>澄清1 </td><td> FisherScientific </td><td> 7401 </td><td>徕卡Autostainer自动</td></tr><tr><td> cytoseal XYL </td><td> FisherScientific </td><td> 8312-4 </td><td>盖玻片mountant </td></tr><tr><td>民建联</td><td>向量</td><td> SK-4100 </td><td> HRP的发色</td></tr><tr><td> Deeb医师</td><td> DAKO公司</td><td> S2003 </td><td>内源性的AP阻断试剂</td></tr><tr><td>曙红Y酒精</td><td> FisherScientific </td><td> 71204 </td><td>徕卡Autostainer自动</td></tr><tr><td>乙醇，100％，95％，90％，70％ </td><td> FisherScientific </td><td>各个</td><td> deparaffinization和盖片</td></tr><tr><td>苏木</td><td> DAKO公司</td><td> S3301 </td><td> Dako公司Autostainer自动</td></tr><tr><td>苏木7211 </td><td> FisherScientific </td><td> 7211 </td><td>徕卡Autostainer自动</td></tr><tr><td>抗体（初级抗体的所有主机的物种） </td><td>各个</td><td>各个</td><td>初选的阴性对照</td></tr><tr><td>液体永固红（LPR）， </td><td> DAKO公司</td><td> K0640 </td><td>美联社发色</td></tr><tr><td> MACH2美联社</td><td>保科医疗</td><td> MALP521L </td><td>山羊抗小鼠AP共轭</td></tr><tr><td> MACH2酶</td><td>保科医疗</td><td> MHRP520L </td><td>羊抗鼠HRP结合物</td></tr><tr><td> MACH2酶</td><td>保科医疗</td><td> RHRP520L </td><td>羊抗鼠HRP结合物</td></tr><tr><td>甲醇</td><td> FisherScientific </td><td>各个</td><td> H 2 O 2稀释剂</td></tr><tr><td>正常山羊血清</td><td>向量</td><td>的S-1000 </td><td>免疫组织化学阻断试剂</td></tr><tr><td>狙击手</td><td>保科医疗</td><td> BS966M </td><td>免疫组织化学阻断试剂</td></tr><tr><td> TBST 20X </td><td> ThermoScientific </td><td> TA-999 - TT </td><td>免疫组化缓冲区</td></tr><tr><td> triology 20X </td><td>细胞马凯</td><td> 920P-06 </td><td>抗原检索</td></tr><tr><td>二甲苯</td><td> FisherScientific </td><td>各个</td><td> deparaffinization和盖片</td></tr></tbody></table> 表1。特定的试剂。 <table border="1"><tbody><tr><td> 初级抗体 </td><td> 宿主物种 </td><td> 供应商 </td><td> 猫。 ＃ </td><td> 评论和抗原检索 </td></tr><tr><td> 石蜡 </td><td></td><td></td><td></td><td></td></tr><tr><td>淀粉酶</td><td>鼠标</td><td>圣克鲁斯</td><td> SC-46657 </td><td>柠檬酸</td></tr><tr><td>淀</td><td>鼠标</td><td> Serotec公司</td><td> MCA11267 </td><td>柠檬酸</td></tr><tr><td>碳酸酐19.9 </td><td>鼠标</td><td> abcam </td><td> ab15146 </td><td></td></tr><tr><td> Caspase-3的，切割</td><td>兔</td><td>细胞信号</td><td> 9961 </td><td>柠檬酸或三部曲</td></tr><tr><td>抗CD20 </td><td>鼠标</td><td> DAKO公司</td><td> M0755 </td><td>柠檬酸或三部曲</td></tr><tr><td> CD3 </td><td>兔</td><td> DAKO公司</td><td> A0452 </td><td>柠檬酸</td></tr><tr><td> CD34的</td><td>鼠标</td><td>细胞信号</td><td> 3569 </td><td>柠檬酸</td></tr><tr><td>的CD4 </td><td>鼠标</td><td> DAKO公司</td><td> M7310 </td><td>柠檬酸</td> &lt;/ TR&gt; <tr><td> CD45的</td><td>鼠标</td><td> DAKO公司</td><td> M0754 </td><td>柠檬酸</td></tr><tr><td> CD68阳性</td><td>鼠标</td><td> DAKO公司</td><td> M0876 </td><td>柠檬酸</td></tr><tr><td> CD8 + </td><td>鼠标</td><td> DAKO公司</td><td> M7103 </td><td>柠檬酸</td></tr><tr><td>嗜铬素A </td><td>兔</td><td> DAKO公司</td><td> A0430 </td><td>柠檬酸</td></tr><tr><td> CK17 </td><td>鼠标</td><td> DAKO公司</td><td> M7046 </td><td>柠檬酸</td></tr><tr><td> CK19， </td><td>鼠标</td><td> DAKO公司</td><td> M0772 </td><td>柠檬酸</td></tr><tr><td> CK7 </td><td>鼠标</td><td> DAKO公司</td><td> M7018 </td><td>柠檬酸</td></tr><tr><td> C-肽</td><td>兔</td><td>细胞信号</td><td> 4593 </td><td>柠檬酸</td></tr><tr><td> FOXP3 </td><td>鼠</td><td> Ë生命科学</td><td> 14-4776-80 </td&gt; <td>柠檬酸</td></tr><tr><td> ghrelin的</td><td>山羊</td><td>圣克鲁斯</td><td> SC-10386 </td><td></td></tr><tr><td> ghrelin的</td><td>兔</td><td> abcam </td><td> ab85104 </td><td></td></tr><tr><td>胰高血糖素</td><td>兔</td><td> DAKO公司</td><td> A0565 </td><td></td></tr><tr><td>胰高血糖素</td><td>鼠标</td><td> abcam </td><td> ab10988 </td><td>柠檬酸</td></tr><tr><td>胰高血糖素</td><td>豚鼠</td><td>巴赫姆</td><td>的T-5037 </td><td>柠檬酸</td></tr><tr><td> GLUT-1 </td><td>鼠标</td><td> abcam </td><td> ab40084 </td><td>三部曲</td></tr><tr><td> GLUT-2 </td><td>兔</td><td>圣克鲁斯</td><td> SC-9117 </td><td>在人类胰岛细胞不表达</td></tr><tr><td>胰岛素</td><td>豚鼠</td><td> DAKO公司</td><td> A0564 </td><td></td></tr> &lt;TR&gt; <td> Ki-67的</td><td>鼠标</td><td> DAKO公司</td><td> M7240 </td><td>柠檬酸</td></tr><tr><td>玛法</td><td>兔</td><td> Novus公司生物</td><td> NB400-137A </td><td>三部曲</td></tr><tr><td>胰多肽</td><td>兔</td><td> Invitrogen公司</td><td> 18-0043 </td><td>柠檬酸</td></tr><tr><td> PDX-1 </td><td>豚鼠</td><td> abcam </td><td> ab47308 </td><td>柠檬酸或三部曲</td></tr><tr><td>胰岛素原</td><td>鼠标</td><td> novocastra </td><td> NCL-proin-1G4 </td><td></td></tr><tr><td>胰岛素原</td><td>鼠标</td><td>发展研究杂交瘤细胞银行</td><td> GS-9A8 </td><td>柠檬酸</td></tr><tr><td> secretagogin </td><td>兔</td><td>西格玛</td><td>自置居所津贴006641 </td><td>柠檬酸</td></tr><tr><td>平滑肌肌动蛋白</td><td>兔</td><td> abcam </td><td> ab5694 &lt;/ TD&gt; <td></td></tr><tr><td>生长抑素</td><td>兔</td><td> DAKO公司</td><td> A0566 </td><td></td></tr><tr><td>突触素</td><td>鼠标</td><td> DAKO公司</td><td> M0776 </td><td>柠檬酸</td></tr><tr><td> 新鲜冰冻 </td><td> 宿主物种 </td><td> 供应商 </td><td> 猫。 ＃ </td><td> 评论 </td></tr><tr><td> CD11b的</td><td>鼠</td><td> abcam </td><td> ab6332 </td><td>修正4％PF </td></tr><tr><td> CD11c的</td><td>兔</td><td> Serotec公司</td><td> AHP1226 </td><td></td></tr><tr><td> CD25表达</td><td>鼠标</td><td> Novus公司生物</td><td>注：600-564 </td><td>用石蜡切片过夜培养</td></tr><tr><td> CD94的</td><td>鼠标</td><td> abcam </td><td> ab61874 </td><td>修正用丙酮</td></tr><tr><td>谷氨酸脱羧酶</td><td>鼠标</td><td>圣克鲁斯</td><td> SC-130569 </td><td>修正4％PF </td></tr><tr><td> HLA - ABC </td><td>鼠标</td><td> DAKO公司</td><td> M0736 </td><td>修正用丙酮或4％PF </td></tr></tbody></table> 表2。小学抗体。 <table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> aperio Scanscope CS </td><td> aperio </td><td></td><td>数字幻灯片扫描仪</td></tr><tr><td> DAKO公司Autostainer自动增强</td><td> DAKO公司</td><td></td><td>免疫组化的污渍</td></tr><tr><td>标签矩阵打印软件</td><td> BioCarta </td><td> LM7UP57 </td><td>幻灯片标签软件</td></tr><tr><td>徕卡加大Autostainer自动</td><td>徕卡</td><td></td><td> ，H＆é污渍</td></tr><tr><td>溢价盖玻片</td><td> FisherScientific </td><td> 12-548-5J </td><td>盖玻片</td></tr><tr><td>频谱信息管理</td><td> aperio </td><td></td><td>扫描仪软件</td></tr><tr><td> superfrost加滑</td><td> FisherScientific </td><td> 12-550-15 </td><td>正电的幻灯片</td></tr><tr><td>蔬菜蒸笼</td><td>布莱克和德克尔</td><td>各个</td><td>抗原检索</td></tr><tr><td>斑马的TLP 3742 </td><td> CDWG </td><td> TLP3742 </td><td>幻灯片标签打印机</td></tr></tbody></table> 表3。特殊用品和设备。

staining protocols for human pancreatic islets

胰岛面积和数字，在成人胰腺内分泌细胞组成的估计各不相同，从几十万到几百万和β的质量范围从500至1500毫克1-3。这个著名的异质性，开发一个标准的加工和染色过程，使胰腺地区进行了明确规定和胰岛细胞的特点，用严格的组织病理学和免疫组化检查。 本系列第1部分中描述的标准化程序处理人体器官捐赠者的胰腺恢复。胰腺被加工成横截面的3个主要地区（头，身，尾）。横截面从胰头进一步划分，如根据大小，按字母顺序和编号表示小节。这种标准化允许的头部区域的一个完整的交叉截面分析包括钩突的区域，其中包含小岛组成主要的胰多肽细胞的尾部区域。 目前的报告，包括本系列的第2部分，介绍了用于强调胰岛内分泌细胞，复制，和T细胞的浸润胰腺石蜡切片连续切片和病理特征的程序。病理学胰腺节的目的是表征两个外分泌腺，胆管，内分泌组件。在的外分泌腺车厢被评为存在胰腺炎（主动或慢性），萎缩，纤维化，脂肪，以及管道系统，特别是在关系到存在的胰腺导管内肿瘤4。评估胰岛形态，大小，密度，内分泌细胞，炎症，纤维化，淀粉样蛋白，并使用H＆E和免疫组化污渍复制或凋亡细胞的存在。 第2部分中描述的最后一个组件是提供彩色幻灯片作为数字化整体幻灯片图像。数字化幻灯片的情况下，在网上创建一个虚拟的生物资料库的病理数据库胰腺地区举办。访问这个网上收集目前提供超过200名医生和1型糖尿病的研究中科学家参与。联机数据库提供了一个快速和完整的数据共享和调查选择石蜡或冰冻连续切片块的手段。

Watch this Scientific Journal Video about Staining Protocols for Human Pancreatic Islets at JoVE.com

Staining Protocols for Human Pancreatic Islets

本视频演示了人体胰腺胰岛细胞用苏木精伊红（H＆E）和免疫​​组化法（IHC）的鉴定程序。从头部，身体，和尾区域胰腺部分是染色都Ĥ＆é和免疫组化确定胰岛内分​​泌（胰岛素，胰高血糖素，和胰多肽）组成，细胞复制（Ki67的）和（H＆E，CD3）炎性浸润。钩突地区的本地化使用胰多肽免疫组化。

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 ...

staining-protocols-for-human-pancreatic-islets

Research

JoVE Journal

Medicine

26.7K 次观看.  University of Florida. 本视频演示了人体胰腺胰岛细胞用苏木精伊红（H＆E）和免疫​​组化法（IHC）的鉴定程序。从头部，身体，和尾区域胰腺部分是染色都Ĥ＆é和免疫组化确定胰岛内分​​泌（胰岛素，胰高血糖素，和胰多肽）组成，细胞复制（Ki67的）和（H＆E，CD3）炎性浸润。钩突地区的本地化使用胰多肽免疫组化。

视频: Staining Protocols for Human Pancreatic Islets

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 ...

科研

医学

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8.
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12.
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor&#x2019;s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

B7-H1（PD-L1）免疫组化染色石蜡的嵌入式幻灯片的胰腺癌组织

B7-H1/PD-L1, a member  of the B7 family of immune-regulatory cell-surface proteins, plays an important  role in the negative regulation of cell-mediated ...

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes 1-3. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes.
Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa.
By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass 4-6. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets 4,7-15. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1). This method has been used previously to identify novel targets that modulate beta cell replication or function 5,6,8,9,16,17.

This protocol allows one to identify factors that modulate functional beta cell mass to find potential therapeutic targets for the treatment of diabetes. The protocol consists of a streamlined method to assess islet replication and beta cell function in isolated rat islets following manipulation of gene expression with adenoviruses.

复制和β细胞功能评估Adenovirally转分离的啮齿动物胰岛

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

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 ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

生物荧光原位模型胰腺癌进展

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

瘦素受体<sup&gt;分贝</sup&gt; 2型糖尿病小鼠模型：胰岛分离和活细胞双光子成像完好胰岛

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

胰岛门静脉移植小鼠模型的研究

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...