体内多重免疫荧光测定，用于体内衰老标志物的空间评估
2026年7月24日
多重免疫荧光使得单一组织切片内多个抗体标记的顺序标记成为可能，以支持对组织结构和细胞群体的空间分析。本文介绍了多重检测衰老相关、结构和免疫标志物的工作流程，包括手动和自动切片准备、迭代成像以及使用数字病理软件进行单细胞分析。
对衰老细胞的空间分析对于理解其在衰老、癌症、免疫和正常组织稳态等过程中的贡献非常重要。衰老细胞的检测依赖于对多个生物标志物的分析。多重免疫荧光成像允许在单一组织切片内高通量分析这些物质。
首先，请确认5微米厚、福马林固定、石蜡嵌入（FFPE）组织切片，最大尺寸为4 x 2厘米见方，是否中央安装在带正电的显微镜载玻片上，并且在无套片载玻片的观察窗口内清晰可见。执行样品准备步骤，可以手动使用脱隐室或自动染色器。将准备好的玻片放入无套滑套套筒夹中。
将顶部和右边对准金属止挡。用双手同时锁住滑套，将塑料凸轮转到关闭的挂锁位置。确认滑套完全平放，且在支架内不会晃动。
放置ClickWell，将腔体插入滑套上方，用双手捏住侧杆以限制压力并防止断裂。通过入口口装入含50%甘油的含50%甘油的装载介质，通过入口口装入卡扣孔。在幻灯片左侧添加条形码标签，无论方向如何，然后进行成像。
对于区域选择和自荧光分析，请打开Mx Workflow版本4204，即多路复用成像采集软件。点击工具选择协议发布者。在多路复用协议发布者弹窗中，输入协议名称。
点击创建设置轮，然后添加下一个方案轮，创建多轮生物标志物标记。点击发布，然后在弹窗中选择确定。获取Cy3通道以获取虚拟H&E图像。
点击幻灯片，然后创建新批次。选择协议名称后，输入幻灯片ID。点击添加幻灯片，然后选择创建批量处理以创建多路复用实验工作流程。打开Cell DIVE采集软件。
点击加载，将载玻片加载到显微镜中。在工具下拉菜单中，点击网格图标定义所需区域，并使用移动工具重新定位已定义区域。接下来，使用绘图工具取消选中不必要的字段，确保每个视野至少有两个相邻的视野，以避免拼接问题，然后点击“保存区域”。
在Mx工作流程窗口中，点击Viewer QC打开Cell DIVE查看器页面，并以10倍放大率可视化组织图像。点击图像校正图标调整图像亮度，点击提交质量控制（Submit QC）在弹窗中选择“通过并重新加载幻灯片”。图片重新加载后，点击“添加投资回报率”来绘制并调整感兴趣的区域。
选择所需的视野，重复投资回报率的加法。接下来，点击“发送到实验室”。加载载玻片，开始20倍自荧光图像采集。
在成像QC菜单中，选择对应特定幻灯片ID的复选框，然后点击“Viewer QC”。放大加载后的图像，扫描失焦区域或伪影。检查每个通道的背景图像，这些图像将从最终生物标志物信号中扣除。点击提交质检，然后选择“通过并退出”生成背景图片。
在连续生物标志染色前，将抗体在室温下以13,000克离心一分钟，以减少伪影。将抗体溶液以1比200稀释的磷酸盐缓冲生理盐水（PBS）制备，含0.3%牛血清白蛋白（BSA）。通过从ClickWell接口移液去除安装介质。
用含0.01%的PBS或PBST洗涤3次，浸泡5分钟。向端口加入350微升抗体溶液，以确保染色室内样品完全覆盖。在室温下孵育一小时。
接着，从ClickWell接口取出抗体溶液。用2毫升PBST洗三次，每次5分钟。加入2毫升安装介质。
将ClickWell支架插入显微镜，加载载玻片即可开始20倍生物标志物成像。使用混合编辑器和图像校正选项进行成像质量检测，并提交图像质量检测评估。从ClickWell接口取出装载介质，用2毫升PBST洗涤三次，浸泡5分钟。
使用前应新鲜准备染料灭活液。取出PBST后，每张玻片加入2毫升染料灭活液。在载玻套中孵育15分钟。
从ClickWell接口取出染料灭活液，用2毫升PBST洗涤5分钟。加入2毫升装载介质，将ClickWell支架插入显微镜中，即可启动自动的圆特异性20倍自发荧光成像。完成后，按照屏幕上指示的质量控制步骤推进工作流程。
根据需要重复染色、染料灭活和多重成像，直到完成。使用HALO软件选择不同通道，以可视化组织核心不同区域标记的定位。点击“分析”并选择“整张图片”，以分析单个标记的定量指标。
点击结果查看分析组织核心中的细胞总数及表达特定标记的细胞子集。与基于自动玻片染色剂的方法相比，基于手动去隐形腔室的方法导致组织伪影的频率更高，特征是组织剥离以及细胞芯的脱落或滑脱。使用数字病理软件对代表性胰腺组织进行的定量分析显示，p16阳性细胞与较低的LB1水平相关，反之亦然，显示出与衰老相关标志模式一致的特征。
Z分数归一化热图展示了p16阳性层蛋白-B1阴性衰老表型在不同组织中的分布，显示肺、肾上腺和垂体中高浓度的衰老细胞，伴随着p16升高和LB1水平降低。相反，阑尾、淋巴结和脾脏衰老率低，LB1高且p16表达低。该方案允许在单张显微镜切片中探测衰老、结构和免疫生物标志物的组合。
通过在单一样本中结合多个标记，可以进行组织结构和衰老细胞分布的空间分析。未来应用包括对多种组织类型的衰老生物标志物进行大规模剖析，以及与空间组学的整合，以更好地解析细胞异质性。
Overview
This article presents a comprehensive protocol for multiplexed immunofluorescence (IF) staining, enabling the simultaneous detection of multiple antibody markers within a single tissue section. The method is particularly suited for profiling senescent cells across diverse human tissue types, allowing for detailed spatial and quantitative analysis that surpasses traditional IF techniques.
Key Study Components
Area of Science
- Cellular senescence
- Immunofluorescence imaging
- Digital pathology
- Tissue microarray analysis
Background
- Traditional IF imaging is limited in the number of markers that can be assessed simultaneously.
- Senescence lacks a universal biomarker; signatures vary by cell type, trigger, and time since induction.
- Multiplexed IF allows for the detection of multiple markers in a single tissue section, improving cell type and structure profiling.
- Spatial relationships between cell types and tissue structures are critical for understanding senescence in situ.
Purpose of Study
- To provide a detailed protocol for multiplexed IF staining and imaging of human tissue samples.
- To enable unbiased detection and spatial analysis of senescent cells using combinatorial biomarker approaches.
- To demonstrate the utility of multiplexed imaging for comprehensive tissue profiling.
Methods Used
- Tissue microarrays from 34 human tissue types were utilized.
- 10-plex immunofluorescence staining targeting structural, immune, and senescence biomarkers.
- Sequential staining, imaging, and dye inactivation cycles to build multiplex datasets.
- Image acquisition with a multiplex fluorescence slide scanner.
- Protocols for both manual and automated slide preparation.
- Single-cell segmentation and spatial analysis using digital pathology software.
Main Results
- Successful multiplexed detection of multiple biomarkers within single tissue sections.
- Assessment of abundance and spatial distribution of senescent cells across diverse tissues.
- Generation of high-content multiplex datasets suitable for advanced spatial analysis.
- Protocols adaptable for both manual and automated workflows.
Conclusions
- Multiplexed IF provides a powerful, unbiased approach for senescent cell detection in situ.
- The described methods facilitate comprehensive spatial and quantitative analysis of tissue samples.
- This protocol serves as a general guide for multiplexed imaging in research and pathology applications.
Frequently Asked Questions
Executive Industry Relevance
Multiplexed immunofluorescence enables high-content spatial profiling of senescence markers across diverse human tissues, addressing the challenge of heterogeneous senescence signatures in drug discovery. This approach enhances predictive confidence in target validation and supports translational continuity by allowing simultaneous assessment of multiple biomarkers within a single tissue section. The method is positioned to improve decision-making at key inflection points in early discovery and preclinical research pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables comprehensive interrogation of senescence-associated pathways using combinatorial biomarker panels.
- Supports biological de-risking by revealing cell-type and context-specific senescence signatures.
- Facilitates predictive confidence in target selection through spatially resolved marker analysis.
Screening & Assay Development
- Prepares validated tissue systems for downstream screening of senescence-modulating compounds.
- Standardizes multiplexed assay workflows for reproducible and quantitative biomarker detection.
- Enables scalable imaging and analysis suitable for high-throughput tissue microarray studies.
Translational & Preclinical Research
- Aligns spatial biomarker data with disease-relevant tissue contexts for translational biomarker development.
- Provides continuity from discovery through preclinical validation by supporting unbiased senescent cell detection.
- De-risks advancement decisions by quantifying abundance and distribution of senescent cells in situ.
Pipeline & Workflow Integration
This multiplexed IF assay integrates into the discovery-to-preclinical continuum by enabling spatially resolved, quantitative analysis of senescence markers in human tissue microarrays.
- Discovery Biology: Supports hypothesis testing and pathway clarification through multi-marker spatial profiling.
- Screening: Delivers reproducible, quantitative outputs for compound evaluation in validated tissue contexts.
- Analytics: Provides high-content imaging datasets and digital pathology outputs for robust statistical comparison.
- Translational Research: Bridges discovery and preclinical phases by aligning biomarker data with disease-relevant tissues.
- Enterprise Reuse: Offers a scalable, adaptable platform for multiplexed imaging across diverse R&D programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in senescence research.
- Operational Value: Standardizes multiplexed imaging workflows for reproducibility and scalability.
- Strategic Value: Improves go/no-go decisions and capital efficiency by enabling robust spatial biomarker analysis.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of senescence-targeted programs.
Implementation Considerations
- Requires expertise in multiplexed immunofluorescence and digital pathology analysis.
- Needs access to multiplex slide scanners and compatible digital pathology software.
- Demands cross-team standardization of staining, imaging, and analysis protocols.
- Adaptable to both manual and automated slide preparation workflows.
- Dependent on tissue quality and marker panel optimization for reliable outputs.
