Name: staining protocols for human pancreatic islets
Uploaded: 2012-05-23T13:00:00
Description: Watch this Scientific Journal Video about Staining Protocols for Human Pancreatic Islets at JoVE.com

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?...

<ol>
	<li>In't Veld, P., Marichal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+anatomy+of+the+human+islet+of+Langerhans.">Microscopic anatomy of the human islet of Langerhans.</a> Adv. Exp. Med. Biol. 654, 1-19 (2010).</li><li>Matveyenko, A. V., Butler, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+beta-cell+mass+and+diabetes+onset.">Relationship between beta-cell mass and diabetes onset.</a> Diabetes Obes. Metab. 10, 23-31 (2008).</li><li>Saisho, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+volumes+in+humans+from+birth+to+age+one+hundred+taking+into+account+sex,+obesity,+and+presence+of+type-2+diabetes.">Pancreas volumes in humans from birth to age one hundred taking into account sex, obesity, and presence of type-2 diabetes.</a> Clin. Anat. 20, 933-942 (2007).</li><li>Hruban, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+intraepithelial+neoplasia:+a+new+nomenclature+and+classification+system+for+pancreatic+duct+lesions.">Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions.</a> Am. J. Surg. Pathol. 25, 579-586 (2001).</li><li>Campbell-Thompson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+adenocarcinoma+patients+with+localised+chronic+severe+pancreatitis+show+an+increased+number+of+single+beta+cells,+without+alterations+in+fractional+insulin+area.">Pancreatic adenocarcinoma patients with localised chronic severe pancreatitis show an increased number of single beta cells, without alterations in fractional insulin area.</a> Diabetologia. 52, 262-270 (2009).</li><li>Meier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-cell+replication+is+the+primary+mechanism+subserving+the+postnatal+expansion+of+beta-cell+mass+in+humans.">Beta-cell replication is the primary mechanism subserving the postnatal expansion of beta-cell mass in humans.</a> Diabetes. 57, 1584-1594 (2008).</li><li>Rowe, P. A., Campbell-Thompson, M. L., Schatz, D. A., Atkinson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pancreas+in+human+type+1+diabetes.">The pancreas in human type 1 diabetes.</a> Semin. Immunopathol. 33, 29-43 (2011).</li><li>Veld, I. n. '. t., P, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulitis+in+human+type+1+diabetes:+The+quest+for+an+elusive+lesion.">Insulitis in human type 1 diabetes: The quest for an elusive lesion.</a> Islets. 3, 131-138 (2011).</li><li>van Belle, T. L., Coppieters, K. T., von Herrath, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+1+diabetes:+etiology,+immunology,+and+therapeutic+strategies.">Type 1 diabetes: etiology, immunology, and therapeutic strategies.</a> Physiol Rev. 91, 79-118 (2011).</li></ol>

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

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 ...

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 ...

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 ...