Name: four-color fluorescence immunohistochemistry of t-cell subpopulations in archival formalin-fixed, paraffin-embedded human oropharyngeal squamous cell carcinoma samples
Uploaded: 2017-07-29T14:00:00.000+00:00
Duration: 7 min 43 s
Description: Watch this Scientific Journal Video about Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinom

Simone Punt得到荷兰癌症协会授予的UL2010-4801的支持。我们要感谢原始论文的所有合作者，这份JoVE协议基于:Emilie A.Dronkers，Marij JP Welters，Renske Goedemans，SenadaKoljenović，Elisabeth Bloemena，Peter JF Snijders，Arko Gorter和Sjoerd van德伯格

患者样本按照"荷兰生物医学科学学会联合会人体组织适当二次使用行为守则"（www.federa.org）中所述的医学伦理指导进行处理。 准备幻灯片<ol><li>在获得医学伦理委员会许可使用切除材料后，从病理科获取切除的组织材料（任何种类的组织）。 注意:对于目前的实验，预处理的肿瘤样品是从1970年至2011年间在莱顿大学医学中心莱顿大学医学中心诊断的原发性口咽肿瘤获得的，如前所述（n = 162） 13选择 。组织的大小仅受到足够量的肿瘤组织的需求的限制，以可靠地在肿瘤内获得4个显微图像。 </li><li>将组织固定在4％缓冲的福尔马林中至少12小时。在ag中去除水分辐射系列乙醇。在二甲苯中洗涤，并使用自动组织处理器将其包埋在石蜡中。 </li><li> 使用切片机在玻片上切割4μm厚的福尔马林固定的石蜡包埋（FFPE）组织切片。使用标准程序将组织块放在微绒毛组织固定器中。 <ol><li>切成薄片，将其放在含有45℃去离子水的水浴表面。使用适合免疫组织化学染色的载玻片通过将载玻片放置在直接位于该部分下方的水中来切出部分。小心地将滑块向上移动一定角度，将部分从水中取出并将其粘贴在滑块上。 </li></ol></li><li>让载玻片在37℃下干燥过夜。 </li><li>将FFPE载玻片完全浸入新鲜的100％二甲苯中，使其均匀脱蜡3次，每次5分钟。在新鲜的100％乙醇，70％乙醇和50％乙醇中浸泡两次，每次5分钟，将载玻片浸没两次。 </li></ol> 2.执行抗原检索<ol><li>在去离子水中洗涤载玻片5分钟。 </li><li>使用微波将预热三乙二胺四乙酸（EDTA）缓冲液（10mM Tris + 1mM EDTA，pH9.0）加热至沸腾温度。通过将载玻片浸入预热的EDTA缓冲液中并将缓冲液在该温度下保持10分钟在微波中进行抗原恢复。 注意:可以使用功率为900 W的微波炉。 </li><li>让幻灯片在缓冲液中冷却2小时。 </li></ol> 3.组织幻灯片<ol><li>用磷酸盐缓冲盐水（PBS）洗涤载玻片两次，每次5分钟。 </li><li>用疏水笔在组织周围绘制一圈，以防止稀释的抗体从载玻片上溅出。用PBS洗一次;这是可选的。 </li><li>用1％w / v牛血清a稀释的第一抗体（抗CD3 1:50，抗FoxP3 1:200和抗IL-17 1:50）孵育组织白蛋白（BSA）在PBS中于室温过夜。 注意:每张幻灯片的体积取决于组织的大小，但通常为50 - 150μL。以与CD3，CD8和FoxP3抗体相同的稀释度（ 即，兔Ig同种型对照抗体，小鼠IgG1同种型对照抗体和山羊）以相同同种型的抗体替代对于阴性对照载玻片具有未知特异性的抗体IgG同种型对照抗体）。 </li><li>用PBS洗涤载玻片三次，每次5分钟。 </li><li> 用针对一级抗体物种和/或同种型（ 即 1:200驴抗山羊IgG Alexa 488，驴抗兔IgG A546和驴抗小鼠A647）的荧光标记的二抗的组合孵育组织，稀释于1％BSA在PBS中在室温下搅拌1小时。 注意:每张幻灯片的体积取决于组织的大小，但通常为50 - 150μL。 <ol><li>后添加二次抗体，通过将它们存放在暗盒中或通过将盒子包裹在铝箔中，尽可能地保持滑片免受光的影响。 </li></ol></li></ol>装载和复制组织幻灯片<ol><li>在PBS中洗涤载玻片三次，每次5分钟。 </li><li>使用含有核反复染料（ 例如 DAPI）的水性防褪色装载介质安装载玻片。尽快（两周内）使用荧光显微镜分析载玻片。将载玻片在4°C的黑暗中保存，直到分析。 </li></ol> 5.分析染色组织幻灯片<ol><li> 使用装备有20 - 25X物镜的荧光显微镜获取每张幻灯片的四张图像。在包含在幻灯片中的整个肿瘤区域的随机位置拍摄图像，以捕获肿瘤的代表性部分。 注意:应使用250X的标准放大倍率。任何荧光o可以使用r激光扫描显微镜，只要它具有可视化所用荧光染料所需的滤光片和激光器即可。 <ol><li>为了可视化Alexa 488，使用最大激发490，最大发射525。对于Alexa 546的可视化，使用最大激发最大值556，排放最大值为573;为了可视化Alexa 647，使用最大激发650，最大发射665;并且为了可视化DAPI，使用激发最大值为358，发射最大值为461。 </li><li>将图像作为图像文件（ 即 jpeg和tiff）保存在用户可用的软件中。 </li></ol></li><li> 在咨询病理学家之后，基于肿瘤上皮与基质细胞的核和细胞形状，区分肿瘤上皮和肿瘤基质区域。 <ol><li>为了确定总基质表面积，在基质细胞（而不是肿瘤细胞）占据的所有区域周围绘制一条线d使用由显微镜制造商提供的图像分析软件的表面积。 <ol><li>点击图片下的"覆盖"标签;选择"封闭多线"形状并点击它。通过点击该区域直到达到第一个点，划分肿瘤上皮和间质区域之间的边界。这提供了该领域的措施。 注意:一旦形状是封闭的，一个数字将旁边的形状包围所选择的图像的表面面积（A = X微米2）出现。 </li></ol></li><li>从总图像表面积（以图像属性提供）减去该表面积，以计算上皮表面积。通过在其周围绘制线排除血管，坏死和自发荧光区域，记录所提供区域的大小并从上皮表面区域减去。 </li><li>在电子表格文件中保存所有表面积号，以计算平均表面积每张幻灯片的细胞数</li><li>使用图像分析软件，通过检查所有四个通道的荧光来确定细胞是单次，双次还是三次阳性。 <ol><li>使用ImageJ计算每个图像中的单，双和三阳性细胞。 <ol><li>下载ImageJ。点击"文件"和"打开"打开图像并选择要分析的图像。右键单击"点工具"（四行中间的一个红色方块），然后选择"多点工具"。双击"多点工具"，通过为每个单元格类型选择不同的计数器编号来计算图像中不同单元格的数量。 注意:计算每种不同细胞类型的所有细胞，如通过存在一种，两种或所有三种荧光颜色所鉴定的。排除DAPI，因为这种核复染素存在于所有细胞中，并用于区分肿瘤细胞与免疫细胞。每个单元格类型都被计数使用不同的计数器号。通过每次选择不同的计数器，在肿瘤上皮和基质区域中分别计数每种细胞类型的细胞数。不要在血管和大部分自身荧光区域内计数细胞。 </li><li>记录电子表格文件中每个图像在每个计数器仓中分析的单元格数。 </li><li>将肿瘤上皮和肿瘤基质的总表面积划分为每种细胞类型的细胞总数。 </li></ol></li></ol></li></ol></li></ol>统计分析注意:所有统计分析应与统计学家讨论，以确保数据质量。对于一般统计，请使用任何医疗统计指南14 。 <ol><li>使用Spearman等级相关rho测试来测试小区频率之间的相关性。 </li><li>测试阳性细胞数量的统计学差异在使用Wilcoxon Mann-Whitney测试的患者组之间。 </li><li>通过使用Kaplan-Meier曲线生成和对数进行基于细胞数的细胞分组，测试细胞数与无病生存之间的相关性（ 即从诊断到局部或远程复发死亡或疾病的时间）排名分析15 。 </li><li>使用Cox比例风险回归分析进行多变量分析。 </li></ol>

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作者声明没有商业或金融利益冲突。

对于所述的方案，最关键的步骤之一是确定所使用的一抗的正确稀释度。标记的二次抗体的稀释度为1:200，如这些特异性Alexa标记的抗体的制造商所推荐的。然后通过连续稀释来确定一抗的稀释度，优选使用两种不同样本的预期组织类型（在这种情况下，OPSCC）。最佳工作稀释度是在没有背景噪声的情况下获得清除信号并且在相同浓度下没有阴性对照的信号的稀释。根据可用的显微镜​​类型和研?...

口咽鳞状细胞癌（OPSCC）是起源于口咽部的鳞状细胞癌的异质组。危险因素包括OPSCC人乳头瘤病毒（HPV）感染和使用酒精和烟草1,2。免疫反应的作用以及如何在临床环境中使用这种作用刚刚开始探索。肿瘤微环境被吸引到癌症部位的免疫细胞渗透。尽管高CD8 +细胞毒性T细胞频率已经与OPSCC患​​者3的改善生存率相关，但其他T细胞亚群（包括Treg和Th17细胞）的作用尚不清楚4 。而Th1和Th17细胞被认为有助于靶向肿瘤细胞的免疫应答，Treg因其抑制其他T细胞活性的能力而众所周知5 。但是，T的存在暂存器已经发现，在不同的肿瘤类型6有利和不利的响应相关。由于并非所有存在于血液中的免疫细胞以相同的程度渗透到肿瘤中，所以研究局部肿瘤微环境提供了针对肿瘤的免疫应答的最可靠的测量。本研究的目的是确定免疫细胞数量和类型与临床结果之间的相关性。我们使用四色荧光IHC成像分析人类OPSCC中各种T细胞亚群的数量和定位。 我们专注于总T淋巴细胞（CD3 + ），Th17细胞和免疫抑制性FoxP3 + Treg，其分化途径与Th17细胞密切相关。 Th17细胞的特征在于CD3和IL-17的组合。细胞因子IL-17也可以由非T细胞产生7 。我们确定了intra-epit的分布在一系列OPSCC病例中的Helic和基质T细胞，Treg，Th17和IL-17 +非T细胞，并分析与患者生存的相关性。多色荧光IHC用于鉴定CD3，Foxp3和IL-17的表达，与DAPI复染色结合。该测定允许容易且清楚地鉴定肿瘤细胞（使用DAPI核染色）和浸润性T细胞群体（使用不同标记物的组合）。样品制备和染色后，使用荧光显微镜和成像软件分离所使用的不同荧光颜色，并确定存在于肿瘤上皮细胞和肿瘤相关基质中的细胞的数量和类型。 用于量化和表型免疫细胞群体的替代测定法是通过肿瘤或外周样品（ 即血液或腹水）的流式细胞术分析或通过飞行时间（CyTOF）分析的细胞计数。使用这个技术，丢失了关于不同细胞类型的定位和相对分布的所有信息。外周样品的使用和分析也不提供关于哪些细胞能够渗透肿瘤微环境的信息。血液和腹水免疫细胞的分析已经示出不以反映肿瘤组织8,9的免疫细胞浸润的表型和频率。 另一个选择是使用明视野显微镜。这种技术优于荧光成像的优点是没有组织自体荧光。虽然一些样品含有更多的自发荧光 - 特别是红细胞，但也包括其他细胞类型，包括嗜中性粒细胞 - 这些区域可以在几乎所有样品的分析中容易地去除。免疫荧光提供了通过使用一组靶向荧光分析一个样品中的多个标记物的优点nt波长。由于缺乏足够数量的标记物和用于特定抗原的商业上可获得的抗体同种型，目前对于明视野显微术来说，这是不可能的。 这里所描述的多色荧光IHC技术已在许多不同的癌症类型和抗体组合被用于研究不同的免疫细胞群，以及肿瘤细胞表达的分子，如人白细胞抗原（HLA）和PD-L1 10，11， 12 。该方案已经使用许多不同类型的样品和抗体建立和验证。

<table><tbody><tr><td>Pathos Delta Ultra Rapid Tissue Processor</td><td>Milestone and Histostar</td><td></td><td>Automated tissue processor</td></tr><tr><td>Histostar</td><td>Thermo Scientific</td><td></td><td>Tissue block embedding machine</td></tr><tr><td>Formaldehyde</td><td>Baker</td><td></td><td></td></tr><tr><td>Xylol</td><td>Merck</td><td></td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td></td><td></td></tr><tr><td>milliQ water</td><td>Elaga Purelab Chorus</td><td></td><td></td></tr><tr><td>Paraffin wax/Paraclean</td><td>Klinipath</td><td>5079A</td><td></td></tr><tr><td>Microtome tissue holder</td><td>Leica</td><td>RM225</td><td></td></tr><tr><td>Flex IHC side</td><td>Dako</td><td></td><td>Tissue slide</td></tr><tr><td>Tris</td><td>Merk-Milipore</td><td>1,083,821,000</td><td></td></tr><tr><td>EDTA</td><td>Baker</td><td>1073</td><td></td></tr><tr><td>PBS</td><td>Bio-Rad</td><td>BUF036A</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A9647</td><td></td></tr><tr><td>Rabbit anti-CD3</td><td>Abcam</td><td>ab828</td><td>Titrate required antibody dilution</td></tr><tr><td>Mouse IgG1 anti-FoxP3</td><td>Abcam</td><td>ab20034</td><td>Titrate required antibody dilution</td></tr><tr><td>Goat IgG anti-IL-17</td><td>R&amp;amp;D Systems</td><td>AF-317-NA</td><td>Titrate required antibody dilution</td></tr><tr><td>Rabbit Ig isotype control antibody</td><td>Abcam</td><td>ab27472</td><td>Use at same final concentration as anti-CD3</td></tr><tr><td>Mouse IgG1 isotype control antibody</td><td>Abcam</td><td>ab91353</td><td>Use at same final concentration as anti-FoxP3</td></tr><tr><td>Goat IgG isotype control antibody</td><td>ThermoFisher Scientific</td><td>02-6202</td><td>Use at same final concentration as anti-IL-17</td></tr><tr><td>Donkey anti-rabbit IgG A546</td><td>ThermoFisher Scientific</td><td>A10040</td><td>Dilute 1:200 in 1% BSA/PBS</td></tr><tr><td>Donkey anti-mouse-A647</td><td>ThermoFisher Scientific</td><td>A31571</td><td>Dilute 1:200 in 1% BSA/PBS</td></tr><tr><td>Donkey anti-goat IgG A488</td><td>ThermoFisher Scientific</td><td>A11055</td><td>Dilute 1:200 in 1% BSA/PBS</td></tr><tr><td>VectaShield containing DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>LSM700 confocal laser scanning microscope&amp;nbsp;</td><td>Zeiss</td><td></td><td></td></tr><tr><td>LCI Plan-Neofluar 25x/0.8 Imm Korr DIC M27 objective</td><td>Zeiss</td><td>420852-9972-720</td><td></td></tr><tr><td>LSM Zen Software</td><td>Zeiss</td><td>version 2009</td><td></td></tr><tr><td>LSM Image Browser</td><td>Zeiss</td><td>version 4.2.0.121</td><td>Available to download at www.zeiss.com/microscopy/int/website/downloads/lsm-image-browser.html.</td></tr><tr><td>SPSS</td><td>IBM Corp.</td><td>version 20.0</td><td></td></tr><tr><td>ImageJ</td><td></td><td>version 1.50i</td><td>Available to download at http://rsb.info.nih.gov/ij.</td></tr></tbody></table>

four-color fluorescence immunohistochemistry of t-cell subpopulations in archival formalin-fixed, paraffin-embedded human oropharyngeal squamous cell carcinoma samples

四色荧光免疫组织化学（IHC）技术是一种量化目标细胞群的方法，同时考虑到其相对分布及其在组织中的定位。该技术已被广泛应用于研究各种肿瘤类型的免疫浸润。肿瘤微环境被吸引到肿瘤部位的免疫细胞浸润。已经发现不同的免疫细胞群体在肿瘤微环境中发挥不同的作用，并且对疾病的结果具有不同的影响。本手册以口咽鳞状细胞癌（OPSCC）为例介绍了多参数荧光IHC的应用。该技术可以扩展到其他组织样本和感兴趣的细胞类型。在本研究中，我们分析了大型OPSCC队列的上皮内和间质隔室（n = 162）。我们专注于总T淋巴细胞（CD3 + ），免疫抑制调节（Tregs， 即 FoxP3 + ）和T辅助细胞17（Th17）细胞（ 即 IL-17 + CD3 + ），使用核复染色体将肿瘤上皮细胞与基质区分开来。发现大量的T细胞与肿瘤内IL-17 +非T细胞数量较少的患者的无病生存率相关。这表明IL-17 +非T细胞可能与OPSCC中的不良免疫反应相关，这与IL-17和癌症患者的存活率差相关。目前，正在开发使用多达7种不同荧光染料的新型多参数荧光IHC技术，并且能够使肿瘤微环境中的免疫细胞更精确地表征和定位。

从1970年至2011年间，莱顿大学医学中心莱顿大学医学中心（Leiden，荷兰莱顿大学医学中心）诊断的一系列FFPE预处理肿瘤样本，如前所述（n = 162）选择，使用所述方案13进行染色。分析每张幻灯片的1到4个随机图像（ 图1 ）。指出一些自体荧光细胞，其可以通过其完整的黄色外观而明确区分。在这种情况下，细胞真正的双重或三重阳?...

Watch this Scientific Journal Video about Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinom

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

多参数荧光免疫组织化学可用于评估肿瘤微环境中免疫细胞群体的数量，相对分布和定位。该手稿描述了使用这种技术来分析口咽癌中的T细胞亚群。

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 ...

four-color-fluorescence-immunohistochemistry-t-cell-subpopulations

Research

JoVE Journal

Cancer Research

9.6K 次观看.  Leiden University Medical Center. 多参数荧光免疫组织化学可用于评估肿瘤微环境中免疫细胞群体的数量，相对分布和定位。该手稿描述了使用这种技术来分析口咽癌中的T细胞亚群。 

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

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 ...

科研

癌症研究

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

多重条形码图像分析，用于石蜡组织样品单细胞分析中的免疫分析和空间映射表征

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

肿瘤微环境的多重免疫荧光和空间图像分析

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

作者聚焦：多重免疫荧光结合空间图像分析用于肿瘤微环境的临床和生物学评估  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

多重免疫荧光结合空间图像分析，用于肿瘤微环境的临床和生物学评估

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...

绘制肿瘤免疫景观图谱：深入的多重 IHC 分析方案

Multiplex Immunohistochemical Analysis of the Spatial Immune Cell Landscape of the Tumor Microenvironment

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. Multiplex immunohistochemistry is a valuable tool to visualize and identify different types of immune cells in tumor tissues while retaining its spatial information. Here we provide detailed protocols to analyze lymphocyte, myeloid, and dendritic cell populations in tissue sections. Starting from cutting formalin-fixed paraffin-embedded sections, automatic multiplex staining procedures on an automated platform, scanning of the slides on a multispectral imaging microscope, to the analysis of images using an in-house-developed machine learning algorithm ImmuNet. These protocols can be applied to a variety of tumor specimens by simply switching tumor markers to analyze immune cells in different compartments of the sample (tumor versus invasive margin) and apply nearest-neighbor analysis. This analysis is not limited to tumor samples but can also be applied to other (non-)pathogenic tissues. Improvements to the equipment and workflow over the past few years have significantly shortened throughput times, which facilitates the future application of this procedure in the diagnostic setting.

作者聚焦：解锁对肿瘤免疫细胞景观的见解

This protocol describes in detail how immune cell characterization of the tumor microenvironment using multiplex immunohistochemistry is carried out.

肿瘤微环境空间免疫细胞景观的多重免疫组织化学分析

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. ...

使用 mIHC 破译肺鳞状癌中的肿瘤微环境

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been considered the leading cause of death in lung cancer patients. The complexity of the tumor microenvironment requires effective methods to understand cell-to-cell relationships in tumor tissues. The multiplex immunohistochemistry (mIHC) technique has become a key tool for inferring the relationship between the expression of proteins upstream and downstream of signaling pathways in tumor tissues and developing clinical diagnoses and treatment plans. mIHC is a multi-label immunofluorescence staining method based on Tyramine Signal Amplification (TSA) technology, which can simultaneously detect multiple target molecules on the same tissue section sample to achieve different protein co-expression and co-localization analysis. In this experimental protocol, paraffin-embedded tissue sections of lung squamous carcinoma of clinical origin were subjected to multiplex immunohistochemical staining. By optimizing the experimental protocol, multiplex immunohistochemical staining of labeled target cells and proteins was achieved, solving the problem of autofluorescence and channel crosstalk in lung tissues. In addition, multiplex immunohistochemical staining is widely used in the experimental validation of tumor-related, high-throughput sequencing, including single-cell sequencing, proteomics, and tissue space sequencing, providing intuitive and visual pathology validation results.

作者聚焦：增强肺癌样本中的多色荧光定位

This experimental protocol describes and optimizes a multiplex immunohistochemistry (IHC) staining method, mainly by optimizing single-channel antibody incubation conditions and adjusting the settings of antibodies and channels to solve the problems of autofluorescence and channel crosstalk in lung cancer tissues of clinical origin.

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 ...

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

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此视频中的章节

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

多重条形码图像分析，用于石蜡组织样品单细胞分析中的免疫分析和空间映射表征

多重免疫荧光结合空间图像分析，用于肿瘤微环境的临床和生物学评估

多重免疫荧光结合空间图像分析，用于肿瘤微环境的临床和生物学评估

多重免疫荧光结合空间图像分析，用于肿瘤微环境的临床和生物学评估

肿瘤微环境空间免疫细胞景观的多重免疫组织化学分析

肿瘤微环境空间免疫细胞景观的多重免疫组织化学分析

肿瘤微环境空间免疫细胞景观的多重免疫组织化学分析

石蜡包埋肺癌组织的多重免疫组化染色

石蜡包埋肺癌组织的多重免疫组化染色