本研究得到了国家自然科学基金（NO.81860413,81960455）、云南省科技厅基金（202001AY070001-080）、云南省教育厅科研基金（2019J1274）的支持。

该研究获得了云南省肿瘤医院/昆明医科大学第三附属医院医学伦理委员会的批准。所有受试者/法定监护人均签署知情同意书。
1. 载玻片准备
<ol><li>使用切片机切割含有原发性肺肿瘤或肺癌脑转移细胞的成对石蜡块切片，厚度为 4 μm。将切片取出到水中并用镊子分离，选择最好的切片并将其粘附在聚赖氨酸涂层的载玻片上。</li>
	<li>将组织载玻片置于65°C的烘箱中30分钟，以增强组织粘附。</li>
	<li>通过三次更换将载玻片浸入二甲苯中，每次持续 10 分钟。</li>
	<li>逐渐降低酒精浓度，将载玻片在100%乙醇中孵育5分钟，在90%乙醇中孵育5分钟，在75%乙醇中孵育5分钟，并在去离子水中孵育3分钟。</li>
</ol>2. 热诱导表位检索 （HIER）
<ol><li>在去离子水中将100x柠檬酸钠缓冲溶液（pH 6.0）稀释至1x（10mM），制备足够的缓冲溶液以完全浸没载玻片。</li>
	<li>将载玻片放入压力锅中，使其在高温（100°C）和压力（~30psi）下2分钟。加热后，让载玻片在蒸馏水中冷却至室温3分钟。</li>
	<li>将一包52g磷酸盐缓冲盐水（PBS;粉末状）溶解在5L去离子水中，用于制备PBS缓冲溶液（pH 7.0）。将载玻片置于PBS缓冲溶液中5分钟，更换3次。</li>
</ol>3.过氧化物酶阻断
<ol><li>用3%过氧化氢覆盖切片，并在室温下孵育10分钟。在PBS中冲洗载玻片，每次3次，每次5分钟。</li>
	<li>使用滤纸将多余的水从纸巾周边吸收。同时，确保组织是湿润的。</li>
</ol>4. 第一轮一抗孵育
<ol><li>制备CD8（兔单克隆抗体，克隆SP16）和CD20（小鼠单克隆抗体，克隆L26）的一抗的工作混合物，在Bond一抗稀释剂中以1：50稀释。</li>
	<li>将抗体复合物加入切片上，并在室温下孵育1小时。</li>
	<li>制备 0.1% 吐温/磷酸盐缓冲盐水溶液：1 mL 吐温在 1 L PBS 缓冲溶液中。</li>
	<li>用 0.1% 吐温/磷酸盐缓冲盐水洗涤切片，每次 3 次，每次 5 分钟。使用滤纸将多余的水从纸巾周边吸走，同时确保纸巾湿润。</li>
</ol>5. 第一轮二抗孵育
<ol><li>制备荧光标记的山羊抗兔抗体（激发（Ex）：495nm）和山羊抗小鼠抗体（Ex：578nm）的混合物，在磷酸盐缓冲盐水中按1：50稀释。山羊抗兔抗体附着在CD8的一抗上，山羊抗小鼠抗体附着在CD20的一抗上。</li>
	<li>滴加二抗混合物，在室温下孵育1小时。</li>
</ol>6. 热诱导表位修复和过氧化物酶阻断
<ol><li>在去离子水中将100x柠檬酸钠（pH 6.0）稀释至1x（10mM），制备足够的缓冲溶液以完全浸没载玻片。</li>
	<li>将载玻片放入高压锅中，并在高温（100°C）和压力（~30psi）下放置1分钟。加热后，将载玻片放入蒸馏水中冷却至室温3分钟。</li>
	<li>按照步骤 3 中的说明执行过氧化物酶阻断。</li>
</ol>7. 第二轮一抗孵育
<ol><li>制备CD3（兔单克隆抗体，克隆SP7）和CK（小鼠单克隆抗体，MX005）一抗的工作混合物，在一抗稀释剂中以1：50稀释。</li>
	<li>将抗体复合物加入切片上，并在室温下孵育1小时。</li>
	<li>用 0.1% 吐温/磷酸盐缓冲盐水洗涤，每次 3 次，每次 5 分钟。使用滤纸将多余的水从纸巾周边吸走，同时确保纸巾湿润。</li>
</ol>8. 第二轮二抗孵育
<ol><li>制备山羊抗兔（Ex：652nm）和山羊抗小鼠抗体（Ex：590nm）混合物，在磷酸盐缓冲盐水中1：50稀释。山羊抗兔抗体附着在CD3的一抗上，山羊抗小鼠抗体附着在CK的一抗上。</li>
	<li>滴加二抗混合物，在室温下孵育1小时。用 0.1% 吐温/磷酸盐缓冲盐水洗涤，每次 3 次，每次 5 分钟。</li>
</ol>9. 自发荧光猝灭和DAPI染色
<ol><li>将试剂（0.15 M / L KMnO4）加入组织切片1分钟。用流水冲洗 5 分钟。 注意：KMnO4 有毒并损害皮肤。处理载玻片时请务必戴上手套。如果液体滴到皮肤上，请用干净的餐巾纸迅速擦拭干净，然后用流动的水冲洗。</li>
	<li>用浓度增加的酒精（70%，90%，100%）干燥载玻片，每种浓度3分钟。</li>
	<li>添加 DAPI 和盖玻片进行多光谱成像。DAPI的量取决于组织大小。添加盖玻片后，确保组织完全被 DAPI 覆盖。</li>
</ol>10. 幻灯片扫描
<ol><li>将载玻片放在托盘上并推动，直到无法进一步推动。</li>
	<li>要启动程序，请双击桌面上的 程序图标 。在启动过程中，将显示模式选择窗口。选择窗口显示两种明场和荧光模式：自动模式和手动模式。在荧光扫描模式下，单击 自动模式。</li>
	<li>将鼠标光标移到 ？ 按钮显示有关设置的信息。单击“ 过滤器设置”>“过滤器通道 ”以选择 “伪颜色”>“通道号”。定义颜色并保存。</li>
	<li>单击“ 例行工作”>“扫描模式”>“全自动”。单击 “例行工作”>“幻灯片名称 ”以定义输出的幻灯片名称。单击 “日常工作”>“通道设置 ”以选择过滤器。</li>
	<li>单击 “常规工作”>“扫描选项” 以确定虚拟幻灯片的最终质量和存储位置。</li>
	<li>单击 “预览 ”进行阈值设置和选择要扫描的范围。</li>
	<li>单击 “硬件>滤镜”>“实时>”自动对焦“>”自动曝光“>”数字增益“>”滴答声“使用手动曝光时间>”滴答声“限制范围>设置电流。</li>
	<li>单击 “例行工作”>“开始扫描”。选择放大倍率为 20 倍或 40 倍。选择 20x 以获得适当的文件大小。MRXS扩展由3D Pannoramic MIDI扫描仪定义。图像扩展也可以更改为 TIFF 图像。</li>
	<li>单击“ 幻灯片查看器”>“多视图工具箱 ”以选择用于共聚焦的图像，然后对齐和集成图像。</li>
</ol>11. 细胞密度的定量评估
<ol><li>使用 Halo 10 扫描仪软件量化肿瘤和基质区域中阳性免疫细胞（CD3+、CD3+CD8+、CD20+）的百分比。验证 CK 染色以确定肿瘤组织。</li>
</ol>

癌症组织中免疫细胞标志物的原位免疫组织化学检测

<ol>
	<li>Wanleenuwat, P., Iwanowski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastases+to+the+central+nervous+system:+Molecular+basis+and+clinical+considerations.">Metastases to the central nervous system: Molecular basis and clinical considerations.</a> J Neurol Sci. 412, 116755 (2020).</li><li>Schoenmaekers, J., Dingemans, A. C., Hendriks, L. E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+imaging+in+early+stage+non-small+cell+lung+cancer:+still+a+controversial+topic.">Brain imaging in early stage non-small cell lung cancer: still a controversial topic.</a> J Thorac Dis. 10, S2168-S2171 (2018).</li><li>Vilari&#241;o, N., Bruna, J., Bosch-Barrera, J., Valiente, M., Nadal, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunotherapy+in+NSCLC+patients+with+brain+metastases.+Understanding+brain+tumor+microenvironment+and+dissecting+outcomes+from+immune+checkpoint+blockade+in+the+clinic.">Immunotherapy in NSCLC patients with brain metastases. Understanding brain tumor microenvironment and dissecting outcomes from immune checkpoint blockade in the clinic.</a> Cancer Treat Rev. 89, 102067 (2020).</li><li>Babar, Q., Saeed, A., Tabish, T. A., Sarwar, M., Thorat, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+tumor+microenvironment:+Potential+strategy+for+cancer+therapeutics.">Targeting the tumor microenvironment: Potential strategy for cancer therapeutics.</a> Biochim Biophys Acta Mol Basis Dis. 1869 (6), 166746 (2023).</li><li>Goldberg, S. 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=Pembrolizumab+for+management+of+patients+with+NSCLC+and+brain+metastases:+long-term+results+and+biomarker+analysis+from+a+non-randomised,+open-label,+phase+2+trial.">Pembrolizumab for management of patients with NSCLC and brain metastases: long-term results and biomarker analysis from a non-randomised, open-label, phase 2 trial.</a> Lancet Oncol. 21 (5), 655-663 (2020).</li><li>Sukswai, N., Khoury, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+Innovations+for+Diagnosis+and+Tissue-Based+Biomarker+Detection.">Immunohistochemistry Innovations for Diagnosis and Tissue-Based Biomarker Detection.</a> Curr Hematol Malig Rep. 14 (5), 368-375 (2019).</li><li>Janardhan, K. S., Jensen, H., Clayton, N. P., Herbert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+in+Investigative+and+Toxicologic+Pathology.">Immunohistochemistry in Investigative and Toxicologic Pathology.</a> Toxicol Pathol. 46 (5), 488-510 (2018).</li><li>Torlakovic, E. E., Nielsen, S., Vyberg, M., Taylor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+controls+under+control:+the+time+is+now+for+immunohistochemistry.">Getting controls under control: the time is now for immunohistochemistry.</a> J Clin Pathol. 68 (11), 879-882 (2015).</li><li>Tan, W. C. 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=Overview+of+multiplex+immunohistochemistry/immunofluorescence+techniques+in+the+era+of+cancer+immunotherapy.">Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy.</a> Cancer Commun (Lond). 40 (4), 135-153 (2020).</li><li>Wong, P. F., 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=Multiplex+quantitative+analysis+of+tumor-infiltrating+lymphocytes+and+immunotherapy+outcome+in+metastatic+melanoma.">Multiplex quantitative analysis of tumor-infiltrating lymphocytes and immunotherapy outcome in metastatic melanoma.</a> Clin Cancer Res. 25 (8), 2442-2449 (2019).</li><li>Sanchez, K., 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=Multiplex+immunofluorescence+to+measure+dynamic+changes+in+tumor-infiltrating+lymphocytes+and+PD-L1+in+early-stage+breast+cancer.">Multiplex immunofluorescence to measure dynamic changes in tumor-infiltrating lymphocytes and PD-L1 in early-stage breast cancer.</a> Breast Cancer Res. 23 (1), 2 (2021).</li><li>Zhang, W., 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=Multiplex+immunohistochemistry+indicates+biomarkers+in+colorectal+cancer.">Multiplex immunohistochemistry indicates biomarkers in colorectal cancer.</a> Neoplasma. 68 (6), 1272-1282 (2021).</li><li>Salameh, S., Nouel, D., Flores, C., Hoops, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+immunohistochemistry+protocol+for+detecting+the+guidance+cue+Netrin-1+in+neural+tissue.">An optimized immunohistochemistry protocol for detecting the guidance cue Netrin-1 in neural tissue.</a> MethodsX. 5, 1-7 (2018).</li><li>McClellan, P., Jacquet, R., Yu, Q., Landis, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+the+immunohistochemical+identification+and+localization+of+Osterix+in+periosteum-wrapped+constructs+for+tissue+engineering+of+bone.">A Method for the immunohistochemical identification and localization of Osterix in periosteum-wrapped constructs for tissue engineering of bone.</a> J Histochem Cytochem. 65 (7), 407-420 (2017).</li><li>Sun, Y., 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=Sudan+black+B+reduces+autofluorescence+in+murine+renal+tissue.">Sudan black B reduces autofluorescence in murine renal tissue.</a> Arch Pathol Lab Med. 135 (10), 1335-1342 (2011).</li><li>Taube, J. 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=The+Society+for+Immunotherapy+of+Cancer+statement+on+best+practices+for+multiplex+immunohistochemistry+(IHC)+and+immunofluorescence+(IF)+staining+and+validation.">The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation.</a> J Immunother Cancer. 8 (1), 000155 (2020).</li><li>Clarke, G. 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=A+novel,+automated+technology+for+multiplex+biomarker+imaging+and+application+to+breast+cancer.">A novel, automated technology for multiplex biomarker imaging and application to breast cancer.</a> Histopathology. 64 (2), 242-255 (2014).</li><li>Oliveira, V. 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=Sudan+Black+B+treatment+reduces+autofluorescence+and+improves+resolution+of+in+situ+hybridization+specific+fluorescent+signals+of+brain+sections.">Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections.</a> Histol Histopathol. 25 (8), 1017-1024 (2010).</li><li>Ahrens, M. J., Dudley, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+pretreatment+of+growth+plate+cartilage+increases+immunofluorescence+sensitivity.">Chemical pretreatment of growth plate cartilage increases immunofluorescence sensitivity.</a> J Histochem Cytochem. 59 (4), 408-418 (2011).</li><li>Zhang, Y., 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=Spectral+characteristics+of+autofluorescence+in+renal+tissue+and+methods+for+reducing+fluorescence+background+in+confocal+laser+scanning+microscopy.">Spectral characteristics of autofluorescence in renal tissue and methods for reducing fluorescence background in confocal laser scanning microscopy.</a> J Fluoresc. 28 (2), 561-572 (2018).</li></ol>

作者声明，他们没有已知的相互竞争的经济利益或个人关系，这些利益或关系可能会影响本文所报告的工作。

我们已经描述了多重环状荧光免疫组化染色的过程。一抗选择是荧光免疫组化测定的一个关键方面，建议使用单克隆抗体以获得更好的特异性和可重复性。为了优化一抗的工作浓度，通过免疫组化实验测试了一系列稀释液。阳性对照（评估靶抗原表达）和阴性对照（无一抗孵育）都是必不可少的，应进行设置。
在该方案中，一抗被稀释并制备为来自不同物种的混合物。荧光?...

脑转移瘤 （BM） 是最常见的中枢神经系统 （CNS） 肿瘤，发生在近一半的非小细胞肺癌 （NSCLC） 病例中，预后不良1。据估计，10%-20% 的 NSCLC 患者在初始诊断时已经患有 BM，大约 40% 的 NSCLC 病例将在治疗过程中发生 BM2。肿瘤微环境 （TME） 与 NSCLC 的发生和 BM 密切相关，包括各种成分，如血管、成纤维细胞、巨噬细胞、细胞外基质 （ECM）、淋巴样、骨髓来源的免疫细胞和信号分子 3,4。微环境免疫细胞在影响癌细胞生长发育方面起着至关重要的作用。脑转移瘤具有许多潜在的治疗靶点，其特征是复杂的免疫微环境和信号传导过程。例如，PD-1 抑制剂作为免疫检查点抑制剂 （ICI） 已显示出对肺癌脑转移 （LCBM） 患者的临床疗效。然而，原发性 NSCLC 和 LCBM5 对 PD-1 治疗的反应频率不同，这表明肿瘤免疫微环境充当关键的 ICI 调节因子。
免疫组化 （IHC） 是生物学、基础医学和病理学领域的宝贵工具6.该检测方法通过抗原-抗体在组织载玻片上的相互作用来可视化抗原表达7。IHC 用于诊断预测标志物、评估预后标志物、指导靶向治疗和探索肿瘤细胞的生物学功能8。然而，传统的 IHC 方法一次只能检测一种生物标志物。为了解决这一局限性，免疫组织化学技术的创新导致了多重荧光免疫组织化学 （mfIHC） 的发展，它允许在同一组织载玻片上同时识别多个蛋白质标记物，包括明场和荧光场9。这一进展提供了对 TME 内基质细胞、免疫细胞和癌细胞之间的细胞组成和分子相互作用的准确分析。
在这项研究中，我们提出了一种多重环状免疫组织化学方案，以分析免疫细胞的空间分布。同时选择兔和大鼠等两种不同物种的一抗进行孵育，然后进行荧光标记的二抗。抗原修复在每轮抗原抗体反应后进行。阻断自发荧光，并使用4'，6-二脒基-2-苯基吲哚（DAPI）对细胞核进行染色。该panel包括CD3、CD8、CD20和CK的顺序检测，细胞根据标记物分类：肿瘤细胞（CK+）、成熟T细胞（CD3+）、细胞毒性T细胞（CD3+CD8+）、B细胞（CD20+）10,11。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.15 mol/L KmnO4</td>
			<td>Maixin Biotechnology Co. Ltd.</td>
			<td>MST-8005</td>
			<td></td>
		</tr>
		<tr>
			<td>100x sodium citrate&#160;</td>
			<td>Maixin Biotechnology Co., Ltd</td>
			<td>MVS-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>3% hydrogen peroxide</td>
			<td>Maixin Biotechnology Co., Ltd</td>
			<td>SP KIT-A1</td>
			<td></td>
		</tr>
		<tr>
			<td>3D Pannoramic MIDI</td>
			<td>3D histech Ltd</td>
			<td>Pannoramic MIDI 1.18</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Abcam</td>
			<td>ab150113</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568&#160;</td>
			<td>Abcam</td>
			<td>ab175701</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594</td>
			<td>Abcam</td>
			<td>ab150116</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647</td>
			<td>Abcam</td>
			<td>ab150079</td>
			<td></td>
		</tr>
		<tr>
			<td>Bond primary antibody diluent</td>
			<td>Lecia</td>
			<td>AR9352</td>
			<td></td>
		</tr>
		<tr>
			<td>CD20</td>
			<td>Maixin Biotechnology Co., Ltd</td>
			<td>kit-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>Maixin Biotechnology Co., Ltd.</td>
			<td>&#160;kit-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8&#160;</td>
			<td>Maixin Biotechnology Co., Ltd</td>
			<td>RMA-0514</td>
			<td></td>
		</tr>
		<tr>
			<td>CK</td>
			<td>Maixin Biotechnology Co. Ltd.</td>
			<td>MAB-0671,</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>sig-ma</td>
			<td>D8417</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Sinopharm Group Chemical reagent Co., LTD</td>
			<td>10009218</td>
			<td></td>
		</tr>
		<tr>
			<td>Histocore Multicut</td>
			<td>lecia</td>
			<td>2245</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS(powder)</td>
			<td>Maixin Biotechnology Co., Ltd</td>
			<td>PBS-0061</td>
			<td></td>
		</tr>
		<tr>
			<td>slide viwer&#160;</td>
			<td>3D histech Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>xylene</td>
			<td>Sinopharm Group Chemical reagent Co., LTD</td>
			<td>10023418</td>
			<td></td>
		</tr>
	</tbody>
</table>

multiplex cyclic fluorescent immunohistochemistry

肿瘤微环境涉及宿主细胞、肿瘤细胞、免疫细胞、基质细胞和脉管系统之间的相互作用。表征和空间组织免疫细胞亚群和靶蛋白对于预后和治疗目的至关重要。这导致了多重免疫组化染色方法的发展。多重荧光免疫组化可同时检测多种标志物，有助于全面了解细胞功能和细胞间相互作用。在本文中，我们描述了多重环荧光免疫组织化学测定的工作流程及其在淋巴细胞亚群定量分析中的应用。多重荧光免疫组化染色遵循与标准免疫组化类似的步骤和试剂，包括抗原修复、环状抗体孵育和福尔马林固定石蜡包埋 （FFPE） 组织载玻片上的染色。在抗原-抗体反应过程中，制备来自不同物种的抗体混合物。优化和验证抗原回收时间和抗体浓度等条件，以提高信噪比。该技术是可重复的，可作为免疫治疗研究和临床应用的宝贵工具。

Brain metastases (BM) represent the most common central nervous system (CNS) tumors, occurring in nearly half of non-small cell lung cancer cases (NSCLC), with a poor prognosis1. An estimated 10%-20% of NSCLC patients already have BM at the time of initial diagnosis, and approximately 40% of NSCLC cases will develop BM during the course of treatment2. The tumor microenvironment (TME) is closely associated with NSCLC occurrence and BM, including various components, such as blood vessels, fibroblasts, macrophages, extracellular matrix (ECM), lymphoid, bone marrow-derived immune cells, and signaling molecules3,4. Microenvironmental immune cells play a crucial role in influencing cancer cell growth and development. Brain metastases present numerous potential treatment targets characterized by complex immunological microenvironments and signaling processes. For instance, PD-1 inhibitors have shown clinical efficacy for patients with lung cancer brain metastasis (LCBM) as an immune-checkpoint inhibitor (ICI). However, the frequency of responses to PD-1 therapy varies between primary NSCLC and LCBM5, suggesting that the tumor immune microenvironment acts as a critical ICI regulator.
Immunohistochemistry (IHC) is an invaluable tool in the fields of biology, foundation medicine, and pathology6. This detection method visualizes antigen expression through the interaction of antigen-antibody on a tissue slide7. IHC is used for diagnosing predictive markers, evaluating prognostic markers, guiding targeted therapies, and exploring the biological functions of tumor cells8. However, the traditional IHC method can only detect one biomarker at a time. To address this limitation, the innovation of immunohistochemical technology has led to the development of multiplex fluorescence immunohistochemistry (mfIHC), which allows for the simultaneous identification of multiple protein markers on the same tissue slide, both in bright field and fluorescent field9. This advancement provides accurate analysis of cell composition and molecular interactions among stromal cells, immune cells, and cancer cells within the TME.
In this study, we present a protocol for multiplex cyclic immunohistochemistry to analyze the spatial distribution of immune cells. Two primary antibodies of different species, such as rabbit and rat, are chosen for incubation simultaneously, followed by fluorescence-labeled secondary antibodies. Antigen retrieval is performed after each round of antigen-antibody reaction. Autofluorescence is blocked, and 4', 6-diamidino-2-phenylindole (DAPI) is used for staining the nuclei. The panel includes sequential detection of CD3, CD8, CD20, and CK, cells are categorized according to the markers: tumor cells (CK+), mature T cells (CD3+), cytotoxic T cells (CD3+CD8+), B cells (CD20+)10,11.

作者聚焦：使用荧光免疫组织化学高效检测癌症组织中的免疫细胞浸润

多重环状荧光免疫组化

The challenge for immunohistochemistry is to choose the species of primary and secondary monoclonal antibodies to avoid cross-reactivity and obtain better specificity and repeatability. Gender decision is necessary to optimize the antibody dilution ratio. Meanwhile, another challenge is to align the localization of and the sub structures.The use of this protocol meet the technical evolution of the distribution of three lymphocytic subpopulations, such as CT three, positive CT three, and CT eight positive and CT 20 positive based on in cell counts and the protein expression in conserv tissues, the level of infiltration can be observed too. In our protocol, the parameter Antibodies and the Fluor secondary antibodies derive from different species at the iso mixture making us a efficient. This measure also shows a specific measure between antibody and antigen.

我们提出了一种在单张载玻片上使用 5 色多重荧光进行环状抗原检测的方案。通过对检测方法的优化，我们可以孵育来自不同物种的两种抗体（图1）。实验程序所需的设备包括压力锅和免疫染色盒（图2A）。
完成测定后，我们在扫描载玻片之前定义四个标记的伪颜色。CD3、CD8、CD20和CK的伪颜色分别为黄色、红色、绿色和青色。细?...

Watch this Scientific Journal Video about Author Spotlight: Efficient Detection of Immune Cell-Infiltration in Cancer Tissues Using Fluorescent Immunohistochemistry at JoVE.com

多重循环免疫组化允许使用重复抗原抗体孵育、图像扫描以及图像比对和整合同时原 位 检测多个标记物。在这里，我们介绍了使用该技术在肺癌和配对脑转移样本中鉴定免疫细胞底物的操作方案。

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and ...

免疫组化面临的挑战是选择一抗和二抗的种类，以避免交叉反应，并获得更好的特异性和可重复性。性别决定对于优化抗体稀释率是必要的。同时，另一个挑战是协调子结构的本地化和子结构。根据细胞计数和保守组织中的蛋白质表达，使用该方案满足三个淋巴细胞亚群分布的技术演变，例如CT 3，CT阳性3，CT8阳性和CT 20阳性，也可以观察到浸润水平。在我们的实验方案中，参数抗体和福陆二抗来自异丙混合物中的不同物种，使我们高效。该测量还显示了抗体和抗原之间的特定测量。

multiplex-cyclic-fluorescent-immunohistochemistry

Research

JoVE Journal

Cancer Research

Multiplex Cyclic Fluorescent Immunohistochemistry

466 次观看.  The Third Affiliated Hospital of Kunming Medical University & Yunnan Cancer Hospital. 免疫组化面临的挑战是选择一抗和二抗的种类，以避免交叉反应，并获得更好的特异性和可重复性。性别决定对于优化抗体稀释率是必要的。同时，另一个挑战是协调子结构的本地化和子结构。根据细胞计数和保守组织中的蛋白质表达，使用该方案满足三个淋巴细胞亚群分布的技术演变，例如CT 3，CT阳性3，CT8阳性和CT 20阳性，也可以观察到浸润水平。在我们的实验方案?...

视频: 作者聚焦：使用荧光免疫组织化学高效检测癌症组织中的免疫细胞浸润

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

Multiplex cyclic immunohistochemistry allows in situ detection of multiple markers simultaneously using repeated antigen-antibody incubation, image scanning, and image alignment and integration. Here, we present the operating protocol for identifying immune cell substrates with this technology in lung cancer and paired brain metastasis samples.