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>

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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, 增加可在同一实验样品上测量的不同结果的数量。虽然在这里对胰岛进行了优化, 但该技术可以应用于其他低丰度材料, 包括那些小块, 易碎的性质, 或粘性样品, 否则将难以处理。这种方法被优化为新鲜固定的胰岛, 并没有被测试其他类型的材料, 如以前冰冻胰岛。这些部分也可能是有用的原位杂交化验, 以检测 DNA 或 RNA。添加额外的凝胶服务两个目的: 最大限度地发挥胰岛收集和保护胰岛丰富的光盘中心在处理/转移过程中的中断。薄凝胶盘固化迅速, 允许缩短脱水和二甲苯渗透步骤, 石蜡嵌入。在平坦的表面形成圆盘, 而不是在二次容器 (离心管或培养板井) 中, 使光盘更容易物理地转移到磁带上进行处理。与椎间盘技术结果相比, 微细技术降低了胰岛的产量和成群的胰岛分布, 虽然非平面组织排列可以产生更多的胰岛含节。
该协议的关键步骤包括组织固定, 形成凝胶盘, 石蜡嵌入, 切片。关于固定, 如 H & E 和胰岛素和胰高血糖素的免疫荧光检查, 在煤灰和福尔马林之间没有发现差异, 或固定时间不等于 10-30 分钟 (未显示)。但是, 应优化固定强度和持续时间以满足最终用户的需要。本议定书所载的主要创新是制备凝胶胰岛盘;关键步骤概述在图 1中。关键步骤包括使用少量的凝胶将组织集中在一个小区域内, 并在平坦的表面形成圆盘, 以便在单个平面中排列组织以进行切片。使用低结合离心管和尖端和摆动斗离心机为所有的旋转提高组织产量;胰岛坚持在固定后的塑料。添加容易可见的珠子协助圆盘形成, 嵌入和切片。在添加凝胶之前, 尽可能多地去除 PBS 是避免稀释琼脂糖的关键, 这会导致凝固不良, 并难以将圆盘转移到纸张上。保持薄光盘完好无损, 而滑动从玻璃幻灯片到纸张可能是一个挑战。如果光盘中出现皱纹, 请允许它返回原来的位置, 添加更多的凝胶, 并重新冷却幻灯片。对于石蜡嵌入, 重要的是把圆盘嵌入平边和平行的切割平面。histotechnologist 经验与切割石蜡切片是必不可少的成功的这一程序, 因为材料集中在领先的边缘块。过度的面对块可能导致样品的损失。当蓝色珠子可见时, 建议立即开始收集部分。
固定强度和持续时间应为最终用户的特定组织和需要进行优化。如果需要, 可以修改每个样本所用的胰岛数量;然而, 减少起始材料产生的部分, 较少的胰岛。可使用此技术生成更厚或更大的光盘;脱水和二甲苯入渗步骤可能需要加长, 并应加以优化。如果结果节只包含部分圆盘形状, 请考虑光盘未与切削表面平行嵌入的可能性。如果获得的部分太少, 技术人员可能在初始块准备过程中丢失了材料。
该方法生成含有固定胰岛组织的切片, 因此只能用于组织学结果。为了获得大量的胰岛每节, 使用 200-250 IEQ 启动材料, 这是一个挑战, 产生一些实验类型。高质量的部分取决于有经验的 histotechnologist。
从单一实验条件中产生的10多节包含许多小岛的能力将允许在同一材料上量化多个结果, 从而提高实验效率。对完整的胰岛进行例行分析可能会在组织异质性方面取得进展, 不仅在细胞层面上, 而且在胰岛水平上, 目前尚不了解这一点。将此技术应用于其他有限丰度的组织样本也可以为其他领域带来好处。

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&#160;</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 &#956;L)</td>
			<td>Genesee Scientific</td>
			<td>24-430</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Binding Tip (200 &#956;L)</td>
			<td>Genesee Scientific</td>
			<td>24-412</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Binding Tip (20 &#956;L)</td>
			<td>Genesee Scientific</td>
			<td>24-404</td>
			<td></td>
		</tr>
		<tr>
			<td>Low Binding Tip (10 &#956;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&#39;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得。增加用于该程序的胰岛数量增加了各节中的胰岛数目。各节中的胰岛结构多样, 大小不一, 支持一种概念, 即可以使用部分而不是盲注于胰岛间差异的技术获得新的和有趣的数据。该方法同样适用于实验室中隔离的啮齿动物胰岛 (老鼠、老鼠) 和 IIDP 装运后被接收的人胰岛。在胰岛介质中, 小鼠胰岛在隔震后恢复后的形态学明显更完整, 具有平滑的圆形表面和紧凑的球形形状 (图 2a)。人胰岛形态学类似, 当嵌入在抵达日或在装运后恢复过夜后, 可能是因为胰岛已经从隔离压力恢复装运前。微细方法产生了一个较小的组织横截面积, 每节的胰岛少, 密集包装的胰岛和珠子 (图 2B)。图 2B中显示的微细图像是我们用这种方法获得的最好的图象;其中一个样本必须送回, 以减少更多的部分, 因为在第一组的部分没有发现小岛。
该技术为组织学和免疫荧光染色生成高质量的胰岛切片 (图 3)。胰岛素和胰高血糖素染色显示了胰岛结构的不同组成和异质性。这些章节清楚地概括了人类、老鼠和大鼠胰岛之间的已知建筑差异, 人胰岛由大量α细胞与β细胞混合而成, 而老鼠和大鼠胰岛则显示出与β细胞核相似的结构。和周围的阿尔法细胞。串行部分被获得;图 3中的面板显示了与 H & E (左) 和免疫荧光 (右) 相同的胰岛的串行部分。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57931/57931fig1.jpg"> 图 1.制作胰岛凝胶盘的关键步骤的说明。凝胶加热到70°c 在一个热块(a)转移到低结合离心管包含胰岛/珠丸(B)。这些小岛被悬浮在温暖的凝胶中, 转移到玻璃滑梯上, 将材料传播到圆盘状(C D)。另外的干净的凝胶被转移到离心管, 然后所有剩余的材料被去除和传播圆周在最初的胰岛/珠包含盘附近在玻璃滑动(E F)。在冰上凝胶后, 圆盘被转移, 平侧向下, 到组织学纸(G),这是折叠和放置在卡带(H)进行处理。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57931/57931fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57931/57931fig2.jpg"> 图 2。凝胶盘石蜡切片含有大量分布良好的胰岛。(A)低放大板 (40X, 3 x 3 图像缝合在一起, 15% 重叠) 的老鼠, 鼠或人胰岛切片染色的 H & E 表明, 许多胰岛是可见的每一个部分。高倍率镶嵌 (200X) 显示有代表性的胰岛。左面板显示在隔离后立即嵌入的小岛 (老鼠, 大鼠) 或装运 (人) 的部分;右面板显示在文化的隔夜恢复后嵌入的胰岛。在40X 图像中看到的淡蓝色圆圈是嵌入和切片过程中可视化的珠子。这些特殊的样本来自 C57BL/6N 鼠 (1 岁) 和 BBDR 鼠 (4 周), 胰岛由导管胶原酶注射和 Ficoll 分离隔离, 以及从 IIDP 收到的人类 T2D 胰岛。(B) 250 IEQ 的人胰岛嵌入了每老离心管技术, 进行比较。低放大板 (40X; 单个 unstitched 图像捕获所有材料) 和高 mag 插入 (200X) 显示代表部分。培养基 RPMI 10%, 青霉素/链霉素, 5.5 毫摩尔葡萄糖。低放大板的刻度条为1000µm;高放大板的刻度条为50µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57931/57931fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57931/57931fig3.jpg"> 图 3.序列胰岛切片的代表性组织化学和免疫荧光染色.小鼠, 鼠, 和人胰岛的部分嵌入后, 夜间文化染色的 H & E 和成像 brightfield 显微镜 (200X; 左)。相邻的部分染色的胰岛素 (红色), 胰高血糖素 (绿色), 安装在含 DAPI 的媒体 (蓝色) 和成像使用荧光显微镜 (200X; 右)。刻度条:50 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57931/57931fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>

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

Nanyang Technological University

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

Murine Pancreatic Islet Isolation

小鼠胰岛隔离

科研

生物学

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

免疫组化：石蜡切片从Vector实验室使用Vectastain ABC试剂盒

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

使用荧光抗体对小鼠表皮石蜡切片的免疫组织化学

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

人类胰岛的分离：第一部分：收集和胰腺组织消化

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

人类胰岛的分离：第二部分：人类胰岛的分离纯化及文化

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

计算机辅助大型胰岛质量，粒度分布和结构的可视化和量化

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

酵母殖民地嵌入方法

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

基于探针实时荧光定量PCR途径的小分子RNA定量检测

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...