作者要感谢华西医院临床病理研究所的成员，他们为高质量的多重免疫荧光和IHC处理提供了技术指导。该协议得到了中国国家自然科学基金（82200078）的支持。

该协议已获得中国四川大学华西医院伦理委员会的指导意见。在华西医院肺癌中心手术时采集肺癌组织样本，并征得每位患者的知情同意书。
1.组织切片准备
<ol><li>FFPE制备<ol><li>将临床组织浸入中性福尔马林溶液中超过72小时。</li>
		<li>使用二甲苯和分级酒精溶液20完成组织脱水的过程。</li>
		<li>以 4 μm 切片厚度进行手动石蜡包埋和组织切片。使用粘附显微镜载玻片确保组织切片不会脱落。</li>
		<li>将载玻片在65°C的烤箱中烘烤2小时，以确保组织和载玻片干燥。在室温下储存在载玻片盒中。</li>
	</ol></li>
	<li>组织切片预处理<ol><li>将组织载玻片在65°C的烘箱中预处理5分钟，然后依次浸入二甲苯溶液中2×15分钟，无水乙醇溶液2×15分钟，90%乙醇溶液10分钟，85%乙醇溶液10分钟，80%乙醇溶液10分钟，75%乙醇溶液10分钟， 然后用消毒水清洗载玻片 3 x 1 分钟。 注意：该实验技术仅适用于FFPE组织，不适用于冷冻或新鲜组织，因为多次高温抗原修复过程会导致组织从载玻片上脱落。</li>
	</ol></li>
</ol>2. 一抗优化
注：常规IHC实验用于确定单个抗体的孵育条件，主要包括抗体浓度和抗原修复条件。请参阅抗体使用说明书中的条件。
<ol><li>使用略高于推荐浓度范围和中间值的抗体浓度。</li>
	<li>根据蛋白表达的位置优化抗原修复缓冲液程序 PH6 和 PH9。对细胞核中表达的蛋白质使用 PH9，对其他蛋白质使用常规（PH6 或 PH9）。</li>
</ol>3. mIHC染色法
注意：蛋白石 mIHC 染色是可用的 mIHC 方法之一。在这个实验性的 5 色方案中，由于每个组织样品需要用四种抗体染色，因此需要四种一抗孵育、二抗孵育和 TSA 信号放大显色孵育以及五种抗原修复体。最后，进行4'6-二脒基-2-苯基吲哚（DAPI）染色和抗荧光爆破剂密封。
<ol><li>主要试剂制备 注意：根据组织载玻片的大小确定要添加的试剂量。使用足够的体积完全覆盖组织切片（通常每张载玻片 50-150 μL）。实验所需的工作溶液制备如下：<ol><li>洗涤缓冲液工作溶液：用双蒸水以1：20稀释20倍洗涤缓冲液，并在室温下储存。</li>
		<li>PH6缓冲液工作溶液：用双蒸水以1：100稀释100x PH6缓冲液。使用前准备并在室温下储存。</li>
		<li>PH9缓冲液工作溶液：用双蒸水以1：50稀释50x PH9缓冲液。使用前准备并在室温下储存。</li>
		<li>聚合物 HRP：仅针对兔和小鼠来源的一抗制备这种即用型溶液。</li>
		<li>荧光团工作溶液：在 75 μL DMSO 中复制每个荧光团粉末（荧光团 780 除外）。在每次操作之前，将荧光团以1：100的1倍扩增稀释剂稀释。丢弃荧光团工作溶液的任何未使用部分。</li>
		<li>荧光团 780 工作溶液：在 75 μL DMSO 中复溶 TSA-DIG，在 300 μL 双蒸水中复溶荧光团粉末 780。在操作之前，在1x扩增稀释剂中以1：100稀释TSA-DIG稀释，并用Ab稀释剂以1：25稀释荧光团780稀释。</li>
		<li>DAPI工作溶液：用双蒸水或PBS以1：50稀释DAPI溶液。使用前准备并在4°C下储存不超过48小时。 注意。在整个多标记荧光染色实验中，仅用双蒸水和新制备的洗涤缓冲液工作溶液洗涤载玻片。组织切片不得与自来水接触;自来水中的污染物会粘附在组织切片上并引起自发荧光。在整个实验过程中，使样品远离光线。</li>
	</ol></li>
	<li>抗原修复<ol><li>将载玻片放入染色和修复盒中，并用PH 6或PH 9缓冲液填充，盖上盖子，并在微波炉中以100%功率加热2×8分钟。 注意： 在加热过程中向维修箱中加入双蒸水，以防止过度蒸发导致切片变干。</li>
		<li>在继续之前，让载玻片在室温下冷却（30-60分钟）。</li>
		<li>用双蒸水洗涤载玻片3×2分钟。使用疏水阻隔笔包围载玻片上的组织部分。</li>
	</ol></li>
	<li>阻塞<ol><li>将组织切片浸入洗涤缓冲液中，用封闭溶液覆盖，并在室温下将载玻片在加湿室中孵育10分钟。</li>
	</ol></li>
	<li>一抗孵育<ol><li>从载玻片中取出封闭溶液，并用一抗工作溶液覆盖组织切片。将载玻片在培养箱中37°C的加湿室中孵育1小时，或在冰箱中4°C孵育过夜。 注意： 不要让载玻片变干。</li>
	</ol></li>
	<li>聚合物HRP介绍<ol><li>将载玻片在洗涤缓冲液工作溶液中洗涤3×2分钟，将聚合物HRP直接滴加到组织切片中，并在室温下孵育15分钟。 注意：对于储存在冰箱中的载玻片，在洗涤前将其在室温下保存约 30 分钟以去除一抗。</li>
	</ol></li>
	<li>酪胺信号放大 （TSA） 生成<ol><li>用洗涤缓冲液工作溶液洗涤载玻片3×2分钟，将荧光团工作溶液直接滴加到组织切片中，并在室温下孵育10分钟。用洗涤缓冲液工作溶液洗涤载玻片3×2分钟。</li>
	</ol></li>
	<li>荧光基团 780 的特殊程序 注意：荧光团 780 很弱，通常在整个实验的颜色匹配方案中排在最后。荧光团780通常不用于该实验方案，但由于其特异性，列出了其实验步骤。<ol><li>TSA-DIG简介<ol><li>用洗涤缓冲液工作溶液洗涤载玻片3×2分钟，将TSA-Drg工作溶液滴加到组织切片中，并在室温下孵育10分钟。</li>
		</ol></li>
		<li>微波处理<ol><li>用洗涤缓冲液工作溶液洗涤载玻片3×2分钟。</li>
			<li>将载玻片放入染色和修复盒中，填充修复溶液，盖上盖子，然后在微波炉中以 2% 功率加热 8 x 100 分钟。在继续之前，让载玻片在室温下冷却（30-60分钟）。</li>
			<li>用双蒸水清洗载玻片3×2分钟。</li>
		</ol></li>
		<li>荧光基团 780 信号生成<ol><li>将切片浸入洗涤缓冲液中，用荧光团780工作溶液覆盖，并将载玻片在室温下在加湿室中孵育1小时。</li>
			<li>用洗涤缓冲液工作溶液洗涤载玻片3×2分钟。 注意：在此步骤之后不要进行微波处理。</li>
		</ol></li>
		<li>使用荧光基团 780 作为整个工作流程的最后一步，然后直接用 DAPI 工作溶液对细胞核进行染色。 注意：如果不使用荧光基团 780，请跳过步骤 3.7。</li>
	</ol></li>
	<li>微波处理 注意：该微波步骤剥离一级-二级 HRP 复合物并重新暴露抗原，从而允许引入下一个一抗。<ol><li>将载玻片放入染色和修复盒中，用PH 6或PH9缓冲液填充，盖上盖子，然后在微波炉中以100%功率加热2×8分钟。</li>
		<li>在继续之前，让载玻片在室温下冷却（30-60分钟）。用双蒸水清洗载玻片3×1分钟。</li>
		<li>将载玻片浸入洗涤缓冲液工作溶液中2分钟。进行下一次抗体孵育。</li>
	</ol></li>
	<li>下一个抗体孵育<ol><li>重复步骤 3.2-3.8（步骤 3.7 除外）。</li>
	</ol></li>
	<li>细胞核染色和组织密封<ol><li>用DAPI工作溶液覆盖组织切片，并将载玻片在室温下在加湿室中孵育5分钟。用双蒸水清洗载玻片3×2分钟。</li>
		<li>从载玻片中除去水滴，向每张载玻片中加入10-20μL抗荧光淬灭剂，并用显微镜盖玻片密封载玻片。</li>
		<li>将完成的载玻片储存在4°C，避光保存1个月以上。 注意： 注意避免气泡。</li>
	</ol></li>
</ol>4.设置阴性对照
<ol><li>像含组织载玻片一样处理阴性对照载玻片，但不添加一抗、二抗、TSA 试剂和 DAPI 等试剂，这些试剂用于在扫描胶片时去除组织自发荧光。</li>
</ol>5. 组织载玻片全自动扫描
注：用于光谱成像的设备是全自动多光谱组织定量分析仪，可以使用参考系统对 5 色载玻片进行成像和分析可视化（参见 材料表）。该系统使用多光谱成像对多个荧光团和组织自发荧光进行定量解混。
<ol><li>手动设置每个荧光通道的曝光时间和扫描程序，并确认仪器已完成组织载玻片的全自动曝光和扫描。 注意： 载玻片表面必须清洁且无水膜。注意密封剂的用量：密封剂过多会导致盖玻片滑落，仪器报告错误。</li>
</ol>6.荧光分析
<ol><li>双击软件（见 材料表），将原始扫描得到的多色荧光图像文件拖拽到软件中，将图像类型设置为荧光。</li>
	<li>单击 “亮度和对比度 ”按钮，然后检查面板上的通道。单击外部，然后在 通道 上选择感兴趣的荧光通道。拖动 Min 显示 和 Max 显示 以调整每个通道的亮度和对比度。</li>
	<li>分析后，确定要保存的视图，单击 “显示幻灯片概览 ”和 “显示光标位置 ”以删除这两个内容，保留比例尺，然后单击 “文件”|”导出快照 |用于导出 png 文件的当前查看器内容 。 注意：必须导出每个荧光通道和合并的图形以备将来分析。</li>
	<li>如需进一步分析靶细胞阳性，请使用细胞鉴定和分割软件21 （参见 材料表）。</li>
</ol>

使用 mIHC 破译肺鳞状癌中的肿瘤微环境

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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=Challenges+and+opportunities+in+the+statistical+analysis+of+multiplex+immunofluorescence+data.">Challenges and opportunities in the statistical analysis of multiplex immunofluorescence data.</a> Cancers. 13 (12), 3031 (2021).</li><li>Mori, H., 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=Characterizing+the+tumor+immune+microenvironment+with+Tyramide-based+multiplex+immunofluorescence.">Characterizing the tumor immune microenvironment with Tyramide-based multiplex immunofluorescence.</a> Journal of Mammary Gland Biology and Neoplasia. 25 (4), 417-432 (2020).</li><li>Mansfield, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+context+in+epigenetics:+quantitative+multicolor+imaging+and+automated+per-cell+analysis+of+miRNAs+and+their+putative+targets.">Cellular context in epigenetics: quantitative multicolor imaging and automated per-cell analysis of miRNAs and their putative targets.</a> Methods. 52 (4), 271-280 (2010).</li></ol>

所有作者声明不存在利益冲突。

mIHC是科学研究领域中不可缺少的实验技术，用于在单个组织切片中对单细胞水平的多种蛋白质标记物进行定量和空间分析，通过关注原始组织背景下的详细组织结构和细胞相互作用，为疾病病理学研究提供直观准确的数据。mIHC技术的广泛采用将需要优化和有效的实验方案。为了解决实验中可能出现的问题，我们提出了以下考虑和解决方案。
首先，根据多色实验中抗原与荧光...

酪胺信号扩增（TSA）已有20多年的历史，是一类利用辣根过氧化物酶（HRP）对靶抗原进行高密度原位标记的检测技术，广泛应用于酶联免疫吸附测定（ELISA）、原位杂交（ISH）、免疫组化（IHC）等生物抗原检测技术。 大幅提高检测信号的灵敏度1.基于 TSA 技术的蛋白石多色染色最近被开发出来并广泛用于多项研究 2,3,4,5。传统的免疫荧光 （IF） 染色为研究人员提供了一种简单的工具，用于检测和比较各种模式生物细胞和组织中蛋白质的分布。它基于抗体/抗原特异性结合，包括直接和间接方法6。直接免疫染色涉及使用荧光团偶联的一抗来对抗目标抗原，从而可以使用荧光显微镜进行直接荧光检测。间接免疫染色方法涉及应用荧光团偶联的二抗来对抗非偶联的一抗6,7。
传统的单标记免疫荧光染色方法只能对组织中的一种、两种或在某些情况下三种抗原进行染色，这是挖掘组织切片中包含的丰富信息的主要限制。定量结果的解释通常取决于目视观察和成像软件（如 ImageJ）的准确定量。存在技术限制，例如抗体种类限制、荧光标记信号弱和荧光染料颜色重叠（表 1）。Opal 多重 IHC （mIHC） 技术基于 TSA 衍生，允许在同一组织切片上对 7-9 种以上抗原进行多重染色和差异标记，对一抗的来源没有限制，但需要针对抗原的相应抗体具有高度特异性。染色程序与普通免疫荧光染色相似，但有两个区别：每轮染色仅使用一种抗体，并添加抗体洗脱步骤。通过非共价键与抗原结合的抗体可以通过微波洗脱去除，但通过共价键结合到抗原表面的 TSA 荧光信号被保留。
用染料标记的活化酪胺 （T） 分子在靶抗原处高度富集，从而可以有效扩增荧光信号。这允许在没有抗体干扰的情况下直接标记抗原，然后在多个染色循环后可以实现多色标记8,9,10（图1）。尽管该技术可为疾病研究提供可靠和准确的图像，但由于需要广泛的优化和设计，创建有用的多重荧光免疫组化 （mfIHC） 染色策略可能既耗时又严格。因此，该多重panel方案已在自动IHC染色机中进行了优化，其染色时间比手动方案更短。任何研究人员都可以直接应用和调整这种方法，用于人福尔马林固定和石蜡包埋 （FFPE） 组织样本的免疫肿瘤学研究11。此外，载玻片制备、抗体优化和多重设计方法将有助于获得代表准确细胞原位相互作用的可靠图像，并缩短手动分析的优化周期12。
mfIHC主要包括图像采集和数据分析。在图像采集方面，需要用专业的光谱成像设备检测多色标记的复合染色样品，以识别各种混合色信号，获得不受组织自发荧光干扰的高信噪比图像。目前的光谱成像设备主要包括光谱共聚焦显微镜和多光谱组织成像系统。多光谱组织成像系统是专为组织切片定量分析而设计的专业成像系统，其最重要的特点是获取图像光谱信息，提供生物组织样品的形态结构和光学映射信息13,14。光谱图像中的任何像素都包含完整的光谱曲线，每种染料（包括自发荧光）都有其相应的特征光谱，从而能够完整记录和准确识别混合和重叠的多标记信号。
在数据分析方面，由于组织样本的形态结构和组成细胞，多色标记样品极其复杂。普通软件无法自动识别不同的组织类型。因此，智能定量组织分析软件用于特定区域抗原表达的定量分析15,16,17,18。
最重要的是，融合多光谱成像和定量病理分析技术的多标记免疫荧光染色具有检测靶点多、染色有效、分析准确等优点，因此可以显著提高组织形态学分析的准确性，以细胞水平分辨率揭示蛋白质之间的空间关系，有助于从组织切片样本中挖掘更丰富、更可靠的信息19 （表1）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD8</td>
			<td>Abcam</td>
			<td>ab237709</td>
			<td>Primary antibody, 1/100, PH9</td>
		</tr>
		<tr>
			<td>Anti-CD68</td>
			<td>Abcam</td>
			<td>ab955</td>
			<td>Primary antibody, 1/300, PH9</td>
		</tr>
		<tr>
			<td>Anti-CK5/6</td>
			<td>Millipore</td>
			<td>MAB1620</td>
			<td>Primary antibody, 1/150, PH9</td>
		</tr>
		<tr>
			<td>Anti-HMGCS1</td>
			<td>GeneTex</td>
			<td>GTX112346</td>
			<td>Primary antibody, 1/300, PH6</td>
		</tr>
		<tr>
			<td>Animal nonimmune serum</td>
			<td>MXB Biotechnologies</td>
			<td>SP KIT-B3</td>
			<td>Antigen blocking</td>
		</tr>
		<tr>
			<td>Fluormount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td>Anti-fluorescent burst</td>
		</tr>
		<tr>
			<td>Opal PolarisTM 7-Color Manual IHC Kit</td>
			<td>Akoya</td>
			<td>NEL861001KT</td>
			<td>Opal mIHC Staining</td>
		</tr>
		<tr>
			<td>Wash Buffer</td>
			<td>Dako</td>
			<td>K8000/K8002/K8007/K8023</td>
			<td>Washing the tissues slides</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HALO</td>
			<td></td>
			<td></td>
			<td>intelligent quantitative tissue analysis software, paid software</td>
		</tr>
		<tr>
			<td>inForm</td>
			<td></td>
			<td></td>
			<td>intelligent quantitative tissue analysis software, paid software</td>
		</tr>
		<tr>
			<td>PerkinElmer Vectra</td>
			<td></td>
			<td></td>
			<td>multispectral tissue imaging systems, fully automatic scanning of tissue slides.</td>
		</tr>
		<tr>
			<td>QuPath 0.3.2</td>
			<td></td>
			<td></td>
			<td>intelligent quantitative tissue analysis software, open source software, used in this experiment.</td>
		</tr>
	</tbody>
</table>

multiplex immunohistochemistry staining for paraffin-embedded lung cancer tissue

肺癌是全世界恶性肿瘤相关发病率和死亡率的主要原因，复杂的肿瘤微环境被认为是肺癌患者死亡的主要原因。肿瘤微环境的复杂性需要有效的方法来了解肿瘤组织中的细胞间关系。多重免疫组化（mIHC）技术已成为推断肿瘤组织信号通路上游和下游蛋白质表达之间关系以及制定临床诊断和治疗计划的关键工具。mIHC是一种基于酪胺信号放大（TSA）技术的多标记免疫荧光染色方法，可同时检测同一组织切片样品上的多个靶分子，实现不同的蛋白质共表达和共定位分析。在该实验方案中，对临床来源的肺鳞状癌的石蜡包埋组织切片进行多重免疫组织化学染色。通过优化实验方案，实现了标记靶细胞和蛋白的多重免疫组织化学染色，解决了肺组织中的自发荧光和通道串扰问题。此外，多重免疫组化染色广泛应用于肿瘤相关、高通量测序的实验验证，包括单细胞测序、蛋白质组学和组织空间测序，提供直观、可视化的病理学验证结果。

优化CD8一抗与荧光基团的配配方案。两组荧光结果对应于实验组中完全相同的抗体孵育条件，只是抗体匹配的荧光团发生了变化。如图2所示，CD8+ T细胞与荧光基团480和荧光基团690的通道匹配存在显著差异。荧光基团690的通道显示出极强的荧光背景——黄色圆圈内的大面积红色不规则荧光显示颜色，这对CD8+ T细胞的定位分析造成很大的干扰（图2C，D<stron...

Watch this Scientific Journal Video about Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue at JoVE.com

作者聚焦：增强肺癌样本中的多色荧光定位

该实验方案描述并优化了一种多重免疫组化（IHC）染色方法，主要通过优化单通道抗体孵育条件和调整抗体和通道的设置来解决临床来源的肺癌组织中的自发荧光和通道串扰问题。

石蜡包埋肺癌组织的多重免疫组化染色

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been ...

我们的研究重点是优化从临床来源获得的石蜡切片的 mIHC 方案。我们旨在有效解决肺组织中的自发荧光和自发荧光通道串扰问题。我们目前面临的实验挑战涉及解决肺组织中的自发荧光和通道串扰问题，从而实现标记靶细胞和蛋白质所需的成功多色荧光定位。我们高效的多抗原原位标记方法，每个抗体只需不到三个小时，无缝集成了多光谱成像系统和专业病理学软件。这种优化的 mIHC 方案很容易适应其他研究实验室的使用。

multiplex-immunohistochemistry-staining-for-paraffin-embedded-lung

Research

JoVE Journal

Cancer Research

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

1.5K 次观看.  Sichuan University. 我们的研究重点是优化从临床来源获得的石蜡切片的 mIHC 方案。我们旨在有效解决肺组织中的自发荧光和自发荧光通道串扰问题。我们目前面临的实验挑战涉及解决肺组织中的自发荧光和通道串扰问题，从而实现标记靶细胞和蛋白质所需的成功多色荧光定位。我们高效的多抗原原位标记方法，每个抗体只需不到三个小时，无缝集成了多光谱成像系统和专业病理学软件。?...

视频: 作者聚焦：增强肺癌样本中的多色荧光定位

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

档案福尔马林固定，石蜡包埋的人类口咽鳞状细胞癌样品中T细胞亚群的四色荧光免疫组织化学

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

科研

癌症研究

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

应用肺转移法研究骨肉瘤转移性肺定植的实用思考

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological information is also necessary. The protocol of immunocytochemical staining on paraffin-embedded cell blocks, presented herein, is an excellent alternative to immunofluorescent staining on non-paraffin-embedded fixed cells. In this protocol, a paraffin cell block from HeLa cells was prepared using the thromboplastin-plasma method, and immunocytochemistry was performed for the evaluation of two proliferation markers, CKAP2 and Ki-67. The nuclei and cytoplasmic morphology of the HeLa cells were well preserved in the cell-block slides. At the same time, the CKAP2 and Ki-67 staining patterns in the immunocytochemistry were quite similar to those in immunohistochemical staining in paraffin cancer tissues. With modified cell-culture conditions, including pre-incubation of HeLa cells under serum-free conditions, the effect could be evaluated while preserving architectural information. In conclusion, immunocytochemistry on paraffin-embedded cell blocks is an excellent alternative to immunofluorescent staining.

Currently, immunofluorescent staining on fixed cells is the method of choice for determination of protein expression levels when morphological information is also necessary. This protocol presented herein provides for an alternative method of immunocytochemistry on paraffin-embedded cell blocks.

石蜡嵌段细胞免疫细胞化学法测定培养体细胞蛋白质表达水平

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

pT3N0M0 胃癌组织石蜡嵌入幻灯片上的弹性染色

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

福尔马林固定癌组织标本免疫肿瘤学自动复合免疫荧光小组研究

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

石蜡包埋三维胶质瘤神经球培养的免疫组织化学评价胶质瘤生物标志物

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of disease processes. The principle techniques used in the past include immunohistochemistry (IHC) and immunofluorescence (IF) which enable visualization of cells as a snapshot in time using between 1 and 4 markers. Both techniques have shortcomings including difficulty staining poorly antigenic targets and limitations related to cross-species reactivity. IHC is reliable and reproducible, but the nature of the chemistry and reliance on the visible light spectrum allows for only a few markers to be used and makes co-localization challenging. Use of IF broadens potential markers but typically relies on frozen tissue due to the extensive tissue autofluorescence following formalin fixation. Flow cytometry, a technique that enables simultaneous labeling of multiple epitopes, abrogates many of the deficiencies of IF and IHC, however, the need to examine cells as a single cell suspension loses the spatial context of cells discarding important biologic relationships. Multiplex fluorescent immunohistochemistry (mfIHC) bridges these technologies allowing for multi-epitope cellular phenotyping in formalin fixed paraffin embedded (FFPE) tissue while preserving the overall microenvironment architecture and spatial relationship of cells within intact undisrupted tissue. High fluorescent intensity fluorophores that covalently bond to the tissue epitope enables multiple applications of primary antibodies without worry of species specific cross-reactivity by secondary antibodies. Although this technology has been proven to produce reliable and accurate images for the study of disease, the process of creating a useful mfIHC staining strategy can be time consuming and exacting due to extensive optimization and design. In order to make robust images that represent accurate cellular interactions in-situ and to mitigate the optimization period for manual analysis, presented here are methods for slide preparation, optimizing antibodies, multiplex design as well as errors commonly encountered during the staining process.

Multiplex fluorescent immunohistochemistry is an emerging technology that enables the visualization of multiple cell types within intact formalin-fixed, paraffin embedded (FFPE) tissue. Presented are guidelines for ensuring a successful 7-color multiplex with instructions for optimizing antibodies and reagents, preparing slides, design and tips for avoiding common problems.

手动复用荧光免疫学的优化、设计和避免陷阱

Microenvironment evaluation of intact tissue for analysis of cell infiltration and spatial organization are essential in understanding the complexity of ...

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide clues to overcoming radioresistance. Voltage-gated calcium channel &#945;2&#948;1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in non-small cell lung cancer (NSCLC) cell lines. Using calcium channel &#945;2&#948;1 subunit as an example of a CSC marker, methods to study the radiosensitivity of CSCs in NSCLC cell lines are presented. CSCs are sorted with putative markers by flow cytometry, and the self-renewal capacity of sorted cells is evaluated by sphere formation assay. Colony formation assay, which determines how many cells lose the ability to generate descendants forming the colony after a certain dose of radiation, is then performed to assess the radiosensitivity of sorted cells. This manuscript provides initial steps for studying the radiosensitivity of CSCs, which establishes the basis for further understanding of the underlying mechanisms.

The presence of cancer stem cells have been associated with relapse or poor outcomes after radiotherapy. This manuscript describes the methods to study the radiosensitivity of cancer stem cells in lung cancer cell lines.

肺癌细胞系中癌症干细胞的放射敏感性

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...