我们感激地感谢麻省糖尿病中心的β细胞生物学小组提供了有益的建议和讨论。人胰岛是由 NIDDK 资助的综合胰岛分布计划 (IIDP) 在希望的城市提供。这项工作由 NIH/NIDDK 资助: R01-DK114686 (LCA), R01-DK105837 (CY), 2UC4DK098085 (IIDP) 和美国糖尿病协会赠款 #1-15 BS-003 (LCA) 与苋菜的顺序合作。

所有涉及动物的程序均由麻省医学院动物保育和使用委员会批准。人胰岛研究是由麻省机构审查委员会决定的, 不符合 IRB 审查或豁免的资格, 因为它们不涉及使用人类科目。
1. 胰岛分离与培养
<ol><li>使用您选择的方法隔离胰岛并分离出污染外分泌物和导管组织。 注意: 该方法是通过胶原酶导管喷洒和 Ficoll 分离12 (啮齿动物) 或装运后胰岛从集成的胰岛分布程序 (IIDP13; 人类) 隔离的胰岛进行优化。小岛被精选了一个 P200 微。</li>
	<li>为了优化胰岛形态学, 允许胰岛在10毫升完整的胰岛培养基 (RPMI 10%, 青霉素/链霉素, 5.5 毫摩尔葡萄糖) 夜间恢复, 在一个湿润室含有 5% CO2在37摄氏度。虽然这一步不需要获得部分, 但在恢复期后, 小岛更完整 (见图 2)。</li>
</ol>2. 胰岛固定
<ol><li>使用低绑定 P200 尖, 精选大约250个胰岛当量 (IEQ)13使用一个校准网格下显微镜到1.5 毫升低结合离心管。低结合提示和管减少胰岛损失。</li>
	<li>允许胰岛沉淀到离心管的底部。删除大多数上清与 P200 吸管提示, 注意不要删除任何胰岛。</li>
	<li>添加1毫升 PBS。离心管在摆动斗离心机, 直到速度达到 200 x g, 然后停止旋转。移除上清。重复 PBS 清洗, 共洗两次。</li>
	<li>添加500µL 10% 福尔马林溶液或4% 新鲜制成的多聚甲醛。在室温下固定30分钟。对于这种方法所测试的结果, 这些固定方法是无法区分的。更短的固定也可能;建议为理想的结果优化固定时间。</li>
	<li>用 P200 提示取出固定剂。</li>
	<li>添加1毫升 PBS。离心管, 直到速度达到 200 x g, 然后停止旋转。移除上清。重复 PBS 清洗, 共洗两次。</li>
	<li>立即执行下一步骤。</li>
</ol>3. 胰岛盘的制备 (图 1)
<ol><li>第一次使用时, 将凝胶 (例如, Histogel) 在70摄氏度处熔化, 并使整除数在1.5 毫升离心管中长期贮存。整除体积并不重要, 因为整除数可以重新使用。</li>
	<li>加热凝胶 (大约100µL 每样品) 在70°c 通过放置离心管包含凝胶整除在热量块设置了到70°c。</li>
	<li>将琼脂糖蓝珠 (每样10µL) 转移到1.5 毫升清洁离心管。在吹打前彻底并用重悬珠子。 注: 蓝珠与胰岛混合, 协助在琼脂糖按钮和石蜡块中对嵌入材料进行可视化识别。使用的蓝色珠子的数量对结果并不重要, 但避免过多的珠子 (> 5x 的胰岛数量), 以允许在各节中最佳地分配胰岛。</li>
	<li>用 1mL PBS 清洗蓝色珠子, 两次。每次清洗时, 在 800 x g 处旋转珠子1分钟。第二次清洗后, 并用重悬珠 (n+ 2) x 10 µL PBS, 其中n是样本管的数量;例如, 对于2样管, 增加到珠子的 PBS 体积将是40µL。</li>
	<li>离心机的离心管包含胰岛 (简要旋转到 200 x g) 和去除大部分上清。用剪刀或刀片切断10µL 微尖的尖端, 并将10µL 的珠子添加到每个胰岛管上, 对每个样品使用干净的尖端。不要混合 (避免失去胰岛)。</li>
	<li>离心机胰岛和珠子 (简单旋转到 200 x g)。删除尽可能多的 PBS 与未切割的10µL 微提示。</li>
	<li>将显微镜幻灯片标记为样本标识。每张幻灯片上都可以准备三个样品。把幻灯片放在热块附近的长凳上, 用温热的凝胶。削减三至五20µL 低绑定微提示每个样品与剃刀刀片或剪刀。加载两个 P20 micropipettes 与削减提示, 以允许快速切换从一个到另一个。</li>
	<li>使用第一微, 加入 15-20 µL 的暖凝胶到胰岛/珠离心管;立即混合, 避免气泡;并将液琼脂/胰岛/珠子混合物应用到滑块中, 形成 < 1 厘米直径的圆盘。将光盘放在幻灯片边缘附近, 为外胶圈留出空间 (下一步)。轻轻轻拍几次, 以解决小岛和珠子。</li>
	<li>使用第二微, 将20µL 的温凝胶添加到样品管中。使用第一微, 混合新的凝胶与任何剩余的胰岛/珠, 重新升温的热量块, 如果它开始凝固, 然后将此混合物应用到幻灯片, 围绕原光盘。根据需要重复使用附加的预切提示, 直到光盘是所需的大小和厚度。 注意: 如果外圈稍厚一点, 处理胶盘就容易多了。通常, 3 x 20 µL 凝胶是充足的。这一步骤最大限度地减少了通过管洗的胰岛的损失, 并添加胰岛可怜的琼脂糖在光盘的外部, 以方便光盘处理。</li>
	<li>如果在每张幻灯片上放置多个光盘, 请单独准备每个光盘并仔细跟踪示例顺序。</li>
	<li>将幻灯片放在湿冰的平坦表面, 盖上盖子, 10 分钟或直到凝胶凝固。如果将光盘从玻璃上滑干, 就更难了。</li>
	<li>标签活检处理/嵌入组织盒与铅笔, 每个样本一个。干燥后的幻灯片, 以避免润湿蓝纸。</li>
	<li>使用刀片的钝边, 轻轻地按每个方向的光盘, 以释放它从玻璃。当它滑动容易, 慢慢地把光盘从显微镜滑到蓝色纸巾纸上。将圆盘, 平边向下, 直接放在纸上。</li>
	<li>保持光盘平整, 折叠纸张周围的光盘, 以防止移动, 将光盘放在折叠的纸在卡带, 并关闭卡带。如果光盘不干净地滑离玻璃, 添加更多的凝胶和/或返回到冰的幻灯片几分钟。</li>
	<li>用烧杯将卡带浸入 PBS 中。石蜡嵌入过程中的最佳形态。</li>
</ol>4. 石蜡嵌入
注: 用下面描述的缩写脱水系列将胰岛凝胶盘加工成石蜡块。此技术是使用自动化处理器进行优化的, 但手动处理应产生类似的结果。
<ol><li>将卡带浸泡在85% 乙醇中15分钟。</li>
	<li>将卡带浸泡在95% 乙醇中15分钟。</li>
	<li>将卡带浸泡在100% 乙醇中15分钟。重复两次, 总共三洗涤。</li>
	<li>将卡带浸泡在二甲苯中15分钟。重复两次, 总共三洗涤。</li>
	<li>将卡带浸泡在熔融石蜡中10分钟。</li>
	<li>将卡带转到新鲜的熔融石蜡中 10-30 分钟。</li>
	<li>小心打开卡带, 拆开蓝纸。取下胶盘, 跟踪与纸张相反的平面。将光盘嵌入到小模具中, 与平面 (含胰岛) 表面平行, 并与切割表面并行。对于插头, 小心地将其从卡带中取出, 并将其嵌入一个小模具中, 尖端指向切割表面。</li>
</ol>5. 石蜡切片和染色
<ol><li>如果需要串行节, 则按顺序标记幻灯片。</li>
	<li>有一个经验丰富的 histotechnologist 切割5µm 石蜡切片。要使产量最大化, 请保存包含材料的所有部分。通常, 收集20个部分可以捕获大部分的材料。</li>
	<li>在室温下贮存部分。切片可用于常规组织学染色 (例如, H & e) 和免疫荧光 (如胰岛素、胰高血糖素和 DAPI)。如有必要, 优化其他抗原的固定。</li>
</ol>

<ol>
	<li>Kim, A., Miller, K., Jo, J., Kilimnik, G., Wojcik, P., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+architecture:+A+comparative+study.">Islet architecture: A comparative study.</a> Islets. 1 (2), 129-136 (2009).</li><li>Steiner, D. J., Kim, A., Miller, K., Hara, 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+islet+plasticity:+Interspecies+comparison+of+islet+architecture+and+composition.">Pancreatic islet plasticity: Interspecies comparison of islet architecture and composition.</a> Islets. 2 (3), 135-145 (2010).</li><li>Campbell-Thompson, M. L., Heiple, T., Montgomery, E., Zhang, L., Schneider, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+protocols+for+human+pancreatic+islets.">Staining protocols for human pancreatic islets.</a> Journal of Visualized Experiments. (63), (2012).</li><li>Da Silva Xavier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cells+of+the+Islets+of+Langerhans.">The Cells of the Islets of Langerhans.</a> Journal of Clinical Medicine. 7 (3), (2018).</li><li>Sharma, R. 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=Insulin+demand+regulates+&#946;+cell+number+via+the+unfolded+protein+response.">Insulin demand regulates &#946; cell number via the unfolded protein response.</a> The Journal of Clinical Investigation. 125 (10), 3831-3846 (2015).</li><li>Stamateris, R. E., 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=Glucose+Induces+Mouse+&#946;-Cell+Proliferation+via+IRS2,+MTOR,+and+Cyclin+D2+but+Not+the+Insulin+Receptor.">Glucose Induces Mouse &#946;-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor.</a> Diabetes. 65 (4), 981-995 (2016).</li><li>Blodgett, D. 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=Novel+observations+from+next-generation+rna+sequencing+of+highly+purified+human+adult+and+fetal+islet+cell+subsets.">Novel observations from next-generation rna sequencing of highly purified human adult and fetal islet cell subsets.</a> Diabetes. 64 (9), 3172-3181 (2015).</li><li>Brissova, 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=Assessment+of+human+pancreatic+islet+architecture+and+composition+by+laser+scanning+confocal+microscopy.">Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy.</a> Journal of Histochemistry &amp; Cytochemistry. 53 (9), 1087-1097 (2005).</li><li>Joiner, K. S., Spangler, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+HistoGelTM-embedded+specimens+for+use+in+veterinary+diagnostic+pathology.">Evaluation of HistoGelTM-embedded specimens for use in veterinary diagnostic pathology.</a> Journal of Veterinary Diagnostic Investigation: Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc. 24 (4), 710-715 (2012).</li><li>Cozar-Castellano, I., Takane, K. K., Bottino, R., Balamurugan, A. N., Stewart, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+beta-cell+proliferation+and+retinoblastoma+protein+phosphorylation+in+rat+and+human+islets+using+adenovirus-mediated+transfer+of+cyclin-dependent+kinase-4+and+cyclin+D1.">Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1.</a> Diabetes. 53 (1), 149-159 (2004).</li><li>La Fortune, K. A., Randolph, M. L., Wu, H. H., Cramer, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+in+cell+block+processing:+The+Cell-Gel+method.">Improvements in cell block processing: The Cell-Gel method.</a> Cancer Cytopathology. 125 (4), 267-276 (2017).</li><li>Alonso, L. 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=Glucose+infusion+in+mice:+A+new+model+to+induce+beta-cell+replication.">Glucose infusion in mice: A new model to induce beta-cell replication.</a> Diabetes. 56 (7), 1792-1801 (2007).</li></ol>

作者没有要申报的利益冲突。

这种改进的基于凝胶盘的嵌入方法提供了一种简单、廉价、高效的方法来生成每节的胰岛高产。在平坦的玻璃表面上构建凝胶盘, 便于在一个明确的区域内均匀分布的小岛传播。将胰岛分布在平面盘中, 可以将许多胰岛放置在切片的平面上, 优化产量, 并允许使用较少的胰岛。圆盘厚度可以调整, 以满足调查人员的需要。由于可以获得多个部分, 同一胰岛的串行部分可以进行分析, 增加可在同一实验?...

胰岛作为循环胰岛素的唯一来源, 是研究糖尿病的研究者的重要组织。从任何给定的有机体, 胰岛有可变大小, 细胞类型频率和建筑学1,2,3。常规的方法研究胰岛的体内结构和内分泌细胞组成是通过切片胰腺组织4,5。由于胰岛只占总胰细胞含量的一小部分, 因此在孤立的胰岛上进行分子研究。体外胰岛培养试验对养分、基因调制 (转染、转导) 或实验性治疗的反应, 为调节内分泌细胞生存、增殖和功能的机制提供重要的洞察力。5,6。
用全胰岛的分子研究或在单层5、6中生长的分散胰岛细胞的组织学或分子研究, 对体外胰岛实验进行分析。整个胰岛的分子分析引入了混合细胞类型的严重警示, 当外推到任何单个细胞类型时, 可能产生假阴性或假阳性结果。细胞分散到盖玻片后培养显微镜允许细胞类型特定的结果测量, 但扰乱胰岛体系结构, 这可能改变对干预的反应, 并排除与建筑有关的结果的识别。此外, 一般只有单一的结果可以测量;例如, 为了在相同的条件下测量β细胞增殖和β细胞死亡, 需要进行两个单独的实验。这些方法也忽视了胰岛间的变异性, 这是该领域越来越感兴趣的领域。用流式细胞仪对胰岛细胞进行细胞型特异分子研究, 或单细胞 RNA 研究是优雅但昂贵, 耗时, 受组织丰度限制, 建筑根除, 不太适合常规细胞培养分析5,7. 全芒 immunostained 岛的共焦成像提供高质量的完整的体系结构数据, 但劳动力密集型, 从每个样本获得的数据仅限于单染色8中可识别的结果。
在后培养的整个胰岛中产生高质量石蜡切片的能力将解决其中的许多问题。高成本, 低丰度的胰岛组织从独特的遗传模型或从人体器官捐献者, 或胰岛状态后的体内或体外实验操作, 是宝贵的。从同一个胰岛获得多个石蜡切片, 将允许来自同一实验的多细胞型特定的、完整的结构分析。
现有的用于切片的胰岛丸的技术是不完善的。组织学优化琼脂糖是一种水低熔点凝胶, 广泛用于处理组织学和细胞学标本, 包括小的或零碎的组织样本难以处理9。一种胰岛嵌入方法是在离心管中悬浮在琼脂糖中的胰岛, 离心机将材料颗粒, 回收琼脂插头, 然后加工和嵌入切片10,11。从管子底部提取凝固试样费时费力, 导致样品偶尔破碎, 人身伤害风险大。胰岛集中在插头的尖端, 导致不充分的胰岛分布在该方法获得的部分。插头的圆形底部使嵌入变得复杂, 这样就可以为切片提供一个小岛穷区域。总体上, 该方法在结果剖面上导致低产量和成群的胰岛分布。
这一新方法是一种简化和改进的方法, 为胰岛切片的准备。胰岛集中在一个小体积, 然后放在显微镜滑动的平滑表面形成一个小圆盘, 与胰岛在一个单一的平面。Histogel 胰岛盘随后被加工成石蜡嵌入在缩短脱水和二甲苯渗透协议。以前的方法, 集中的胰岛在离心管底部, 也进行了比较。这一新的技术提高了每节胰岛的产量, 每个部分的胰岛分布, 并花费较少的时间把胰岛块转移到磁带盒。这项技术是有用的胰岛生物学家或其他科学家研究的小块组织希望通过测量一个单一的样本在其本机组织结构的多个结果, 以最大限度地利用低丰度组织的生产。

<table><tbody><tr><td>1.5 mL Eppendorf tube</td><td>Norgen Bioteck Corp</td><td>P/N 10113&amp;nbsp;</td><td></td></tr><tr><td>Ranin Classic Starter Kit</td><td>ShopRanin</td><td>17008708</td><td></td></tr><tr><td>Ranin ClassicPipette PR-2</td><td>ShopRanin</td><td>17008648</td><td></td></tr><tr><td>Low Binding Tip (1000 &amp;mu;L)</td><td>Genesee Scientific</td><td>24-430</td><td></td></tr><tr><td>Low Binding Tip (200 &amp;mu;L)</td><td>Genesee Scientific</td><td>24-412</td><td></td></tr><tr><td>Low Binding Tip (20 &amp;mu;L)</td><td>Genesee Scientific</td><td>24-404</td><td></td></tr><tr><td>Low Binding Tip (10 &amp;mu;L)</td><td>Genesee Scientific</td><td>24-401</td><td></td></tr><tr><td>10% Formalin solution</td><td>Sigma</td><td>HT501128</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710-S</td><td></td></tr><tr><td>Heatblock</td><td>VWR</td><td>949312</td><td></td></tr><tr><td>HistoGel</td><td>Thermo Scientific</td><td>HG-4000-012</td><td></td></tr><tr><td>Agarose blue beads- Affi-gel</td><td>Bio-Rad</td><td>153-7301</td><td></td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Life technologies</td><td>14190-144</td><td></td></tr><tr><td>Tissue processing cassette</td><td>Simport</td><td>M492-10</td><td></td></tr><tr><td>Bio-Wraps</td><td>Leica-Surgipath</td><td>3801090</td><td></td></tr><tr><td>Citadel 2000 tissue processor</td><td>Thermo-Shandon LLC</td><td></td><td></td></tr><tr><td>Ethanol 200 proof</td><td>Decon Laboratories, INC</td><td>2701</td><td></td></tr><tr><td>Xylene</td><td>Fisher Chemical</td><td>X5SK-4</td><td></td></tr><tr><td>Paraffin</td><td>McCormick Scientific</td><td>39503002</td><td></td></tr><tr><td>Microtome</td><td>Thermo-Shandon LLC</td><td>Finesse ME+</td><td></td></tr><tr><td>Insulin antibody</td><td>DAKO</td><td>A0564</td><td></td></tr><tr><td>Glucagon antibody</td><td>Sigma</td><td>G2654</td><td></td></tr><tr><td>Fluoroshield with DAPI</td><td>Sigma</td><td>F6057</td><td></td></tr><tr><td>Alex fluor 594 secondary antibodies</td><td>Life technologies</td><td>A11076</td><td></td></tr><tr><td>Alex fluor 488 secondary antibodies</td><td>Life technologies</td><td>A11001</td><td></td></tr></tbody></table>

pancreatic islet embedding for paraffin sections

使用孤立胰岛的实验对糖尿病研究很重要, 但胰岛价格昂贵, 丰度有限。胰岛在结构化结构中含有混合细胞种群, 影响功能, 而人胰岛在细胞类型组成中有广泛的变化。目前常用的研究培养胰岛的方法包括在整个胰岛上进行的分子研究, 将不同的胰岛细胞类型聚集在一起, 或对分散的胰岛细胞进行显微或分子研究, 扰乱胰岛结构。在体内胰岛研究中, 石蜡嵌入胰腺切片是一种强有力的技术来评估细胞特异的结果, 在当地的胰腺环境。通过石蜡切片研究后培养胰岛可以提供以下几个优点: 在同一个胰岛上检测多个结果 (可能甚至是同一个胰岛, 使用串行切片), 细胞类型特定测量, 以及维护本机胰岛细胞和细胞基质相互作用在实验性接触和分析。然而, 现有的嵌入孤立的胰岛后培养技术是低效的, 耗时的, 容易丢失的材料, 并通常产生的部分有不足的胰岛数字是有用的量化结果。临床病理实验室细胞块准备设施是不可访问的和不切实际的基础研究实验室。我们开发了一种改进的、简化的工作台顶部方法, 它产生的部分具有良好的产量和小岛分布。固定胰岛是悬浮在温暖的组织学琼脂糖凝胶和 pipetted 成一个平盘上的标准玻璃幻灯片, 这样的小岛分布在飞机上。在标准脱水和嵌入后, 可以从同一个胰岛块中切割出多个 (10 +) 4-5 µm 节。利用这种方法, 可以对小鼠、大老鼠和人胰岛进行组织学和免疫荧光分析。这是一个有效的, 廉价的, 节省时间的方法, 以评估细胞类型特定的, 完整的体系结构的结果, 从培养的胰岛。

图 1显示了准备凝胶盘的步骤的图解示意图。这种凝胶盘法导致石蜡切片, 其中含有足够数量的胰岛分布在一个单一的平面, 以允许有意义的量化的结果。图 2显示了结果节的低放大图像, 以说明每节捕获的胰岛的数量。一般情况下, > 35 个胰岛是可见的每一个部分, 当 250 IEQ 被用于该程序, 和 > 10 节 (4-5 µm) 包含胰岛从每个样本获?...

Watch this Scientific Journal Video about Pancreatic Islet Embedding for Paraffin Sections at JoVE.com

Pancreatic Islet Embedding for Paraffin Sections

体外胰岛研究对糖尿病研究具有重要意义。现有的技术, 研究培养的胰岛在其本机3维结构是耗时, 效率低下, 很少使用。这项工作描述了一种新的, 简单, 有效的方法, 以产生高质量的整个培养胰岛石蜡切片。

胰岛嵌段石蜡切片

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

pancreatic-islet-embedding-for-paraffin-sections

Research

JoVE Journal

Biology

12.7K 次观看.  UMass Medical School. 体外胰岛研究对糖尿病研究具有重要意义。现有的技术, 研究培养的胰岛在其本机3维结构是耗时, 效率低下, 很少使用。这项工作描述了一种新的, 简单, 有效的方法, 以产生高质量的整个培养胰岛石蜡切片。

视频: Pancreatic Islet Embedding for Paraffin Sections

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 ...

科研

医学

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 ...

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 ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

发育生物学

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

人类胰岛神经网络的高分辨率3D 成像

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

生物工程

The Cutting and Floating Method for Paraffin-embedded Tissue for Sectioning

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial companies have devised complex section transfer systems using fluid water. To simplify this technology, we created a simple method using homemade equipment that combines cutting and floating within a simple thermostatic chamber; therefore, the sections automatically enter the water bath on the water surface. The hippocampus from adult mouse brains, adult mouse kidneys, embryonic mouse brains, and adult zebrafish eyes were cut using both conventional paraffin sectioning and the presented method for comparison. Statistical analysis shows that our improved method saved time and produced higher quality sections. In addition, paraffin sectioning of a whole specimen in a short time is easy for junior operators.

Here, we present a protocol to improve paraffin sectioning. This method combines cutting and floating using a simple thermostatic chamber to avoid the transfer process required by the conventional method. As a result, the efficiency and the number of intact paraffin sections were greatly improved.

切片中石蜡嵌入组织的切割和漂浮方法

Sectioning of the paraffin-embedded tissue is widely used in histology and pathology. However, it is tedious. To improve this method, several commercial ...

神经科学

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared from live human and mouse pancreatic tissue embedded in agarose and cut using a vibratome. This method allows for the tissue to maintain viability and function in addition to preserving underlying pathologies such as type 1 (T1D) and type 2 diabetes (T2D). The slice method enables new directions in the study of the pancreas through the maintenance of the complex structures and various intercellular interactions that comprise the endocrine and exocrine tissues of the pancreas. This protocol demonstrates how to perform staining and time-lapse microscopy of live endogenous immune cells within pancreatic slices along with assessments of islet physiology. Further, this approach can be refined to discern immune cell populations specific for islet cell antigens using major histocompatibility complex-multimer reagents.

This study presents the application of live pancreatic tissue slices to the study of islet physiology and islet-immune cell interactions.

观察活胰腺组织切片中的胰岛功能和胰岛-免疫细胞相互作用

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared ...

免疫与感染

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 ...

通过腹腔注射药物诱导小鼠胰腺损伤

Preparing a Mice Model of Severe Acute Pancreatitis via a Combination of Caerulein and Lipopolysaccharide Intraperitoneal Injection

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological changes associated with SAP using animal models can aid in identifying potential therapeutic targets and exploring novel treatment approaches. Previous studies primarily induced pancreatic injury through retrograde bile duct injection of sodium taviaurocholate, but the impact of surgical damage on the quality of the animal model remains unclear. In this study, we employed various frequencies of intraperitoneal Caerulein injections combined with different doses of LPS to induce pancreatic injury in C57BL/6J mice and compared the extent of injury across five intraperitoneal injection protocols. Regarding inducing acute pancreatitis in mice, an intraperitoneal injection protocol is proposed that results in a mortality rate as high as 80% within 5 days. Specifically, mice received ten daily intraperitoneal injections of Caerulein (50 &#181;g/kg), followed by an injection of LPS (15 mg/kg) one hour after the last Caerulein administration. By adjusting the frequency and dosage of injected medications, one can manipulate the severity of pancreatic injury effectively. This model exhibits strong controllability and has a short replication cycle, making it feasible for completion by a single researcher without requiring expensive equipment. It conveniently and accurately simulates key disease characteristics observed in human SAP while demonstrating a high degree of reproducibility.

作者聚焦:确定开发小鼠严重急性胰腺炎新疗法的治疗靶点

Intraperitoneal drug administration is a safe and effective non-invasive approach for inducing pancreatic injury. This study compared five distinct intraperitoneal injection protocols on mice to induce varying degrees of pancreatic injury and established a model of severe pancreatic injury to investigate the pathological changes and treatment strategies for severe acute pancreatitis (SAP).

通过 Caerulein 和 Lipopolysaccharide 联合腹腔注射制备严重急性胰腺炎小鼠模型

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological ...

胰岛嵌段石蜡切片

摘要

探索更多视频

人体胰腺的收集协议

人类胰岛染色协议

从手术标本的人类胰岛细胞激光显微切割的改进方法

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

人类胰岛神经网络的高分辨率3D 成像

切片中石蜡嵌入组织的切割和漂浮方法

观察活胰腺组织切片中的胰岛功能和胰岛-免疫细胞相互作用

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

通过 Caerulein 和 Lipopolysaccharide 联合腹腔注射制备严重急性胰腺炎小鼠模型

通过 Caerulein 和 Lipopolysaccharide 联合腹腔注射制备严重急性胰腺炎小鼠模型