optimizing multiplex immunohistochemistry (mihc) staining for lung cancer tissue samples to address autofluorescence issues

使用 mIHC 破译肺鳞状癌中的肿瘤微环境

Tyramine signal amplification (TSA), which has a history of more than 20 years, is a class of assay techniques that use horseradish peroxidase (HRP) for high-density in situ labeling of target antigens and is widely applied in enzyme-linked immunosorbent assays (ELISAs), in situ hybridization (ISH), immunohistochemistry (IHC), and other techniques for the detection of biological antigens, substantially improving the sensitivity of the detected signal1. Opal polychromatic staining based on TSA technology has been recently developed and widely used in several studies2,3,4,5. Traditional immunofluorescence (IF) staining provides researchers with an easy tool for the detection and comparison of the distribution of proteins in the cells and tissues of various model organisms. It is based on antibody-/antigen-specific binding and includes direct and indirect approaches6. Direct immunostaining involves the use of a fluorophore-conjugated primary antibody against the antigen of interest, which enables direct fluorescent detection using a fluorescence microscope. The indirect immunostaining approach involves the application of a fluorophore-conjugated secondary antibody against the unconjugated primary antibody6,7.
Traditional, single-label, immunofluorescence staining methods can stain only one, two, or in some cases, three antigens in tissues, which is a major limitation in mining the rich information contained in tissue sections. The interpretation of quantitative results often depends on visual observation and accurate quantification by imaging software, such as ImageJ. There are technical limitations, such as antibody species restriction, weak fluorescent label signals, and fluorescent dye color overlap (Table 1). The Opal multiplex IHC (mIHC) technique is based on TSA derivation, which allows multiplex staining and differential labeling of more than 7-9 antigens on the same tissue section, with no restriction on the origin of the primary antibody, but requires high specificity of the corresponding antibody against the antigen. The staining procedure is similar to that of normal immunofluorescence staining, except for two differences: each round of staining involves the use of only one antibody and an antibody elution step is added. Antibodies bound to the antigen by noncovalent bonds can be removed by microwave elution, but the TSA fluorescent signal bound to the surface of the antigen by covalent bonds is retained.
Activated tyramine (T) molecules labeled with the dye are highly enriched at the target antigen, allowing efficient amplification of the fluorescent signal. This allows direct labeling of the antigen without antibody interference, and then multicolor labeling can be achieved after multiple staining cycles8,9,10 (Figure 1). Although this technology produces reliable and accurate images for the study of disease, the creation of a useful multiplex fluorescent immunohistochemistry (mfIHC) staining strategy can be time-consuming and exacting due to the need for extensive optimization and design. Therefore, this multiplex panel protocol has been optimized in an automated IHC stainer with a shorter staining time than that of the manual protocol. This approach can be directly applied and adapted by any researcher for immuno-oncology studies on human formalin-fixed and paraffin-embedded (FFPE) tissue samples11. Moreover, the methods for slide preparation, antibody optimization, and multiplex design will be helpful in obtaining robust images that represent accurate cellular interactions in situ and to shorten the optimization period for manual analysis12.
The mfIHC mainly includes image acquisition and data analysis. In terms of image acquisition, multicolor-labeled complex-stained samples need to be detected with professional spectral imaging equipment to identify the various mixed color signals and obtain high signal-to-noise images without interference from tissue autofluorescence. Current equipment for spectral imaging mainly includes spectral confocal microscopes and multispectral tissue imaging systems. The multispectral tissue imaging system is a professional imaging system designed for the quantitative analysis of tissue sections, and its most important feature is the acquisition of image spectral information, which provides both morphological structure and optical mapping information of biological tissue samples13,14. Any pixel in the spectral image contains a complete spectral curve, and each dye (including autofluorescence) has its corresponding characteristic spectrum, which enables the complete recording and accurate identification of mixed and overlapping multilabel signals.
In terms of data analysis, multicolor-labeled samples are extremely complex due to the morphological structure and constituent cells of the tissue samples. Ordinary software cannot automatically identify different tissue types. Hence, intelligent quantitative tissue analysis software is used for quantitative analysis of antigen expression in specific regions15,16,17,18.
Above all, multilabel immunofluorescence staining fused with multispectral imaging and quantitative pathology analysis technology has the advantages of a large number of detection targets, effective staining, and accurate analysis, and it can therefore significantly improve the accuracy of histomorphological analysis, reveal the spatial relationship between proteins with cellular-level resolution, and help to mine richer and more reliable information from tissue section samples19 (Table 1).

作者聚焦：增强肺癌样本中的多色荧光定位

石蜡包埋肺癌组织的多重免疫组化染色

Our research focused on optimizing the mIHC protocol on paraffin section obtained from clinic sources. We aimed to effectively address autofluorescence and autofluorescence channel crosstalk issues in lung tissue. The current experimental challenges we are facing involve addressing the issues of autofluorescence and channel crosstalk in lung tissue, whereby achieving successful multicolor fluorescence localization they need for labeled target cells and proteins.Our highly efficient multiple antigen in situ labeling method, which requires less than three hours per antibody, seamlessly integrates a multispectral imaging system and a specialized pathology software. This optimized mIHC protocol is easily adaptable for use in other research laboratories.

优化肺癌组织样本的多重免疫组化 （mIHC） 染色以解决自发荧光问题

首先，获得先前制备的配方和固定和石蜡包埋的肺癌组织样品载玻片。在 65 摄氏度下孵育 5 分钟。通过将载玻片浸入二甲苯中，然后浸入浓度降低的乙醇溶液中进行脱水。然后将载玻片浸入灭菌水中三次，每次一分钟。对于抗原修复，将载玻片放入染色和修复盒中。用pH 6或9的免疫组化抗原修复缓冲液工作溶液填充。盖上盖子，在微波炉中以 100% 的功率加热八分钟。然后将载玻片冷却至室温。用封闭溶液覆盖组织，然后在室温下在加湿室中孵育10分钟。取出封闭溶液后，用一抗工作溶液覆盖载玻片，并将其置于加湿室中。然后在 37 摄氏度下将该腔室孵育一小时。接下来，用洗涤缓冲液工作溶液洗涤载玻片两分钟。将聚合物辣根过氧化物酶直接滴加到载玻片上，并在室温下孵育15分钟。洗涤载玻片后，将荧光团工作溶液直接滴加到载玻片上，并在室温下孵育10分钟。洗涤载玻片后，如前所述进行微波处理。当载玻片冷却至室温时，用双蒸水洗涤三次，每次一分钟。接下来，将载玻片浸入洗涤缓冲液工作溶液中两分钟，并如图所示进行抗体孵育，辣根过氧化物酶和荧光基团添加。对于细胞核染色，用DAPI工作溶液覆盖载玻片，并在室温下在加湿室中孵育五分钟。然后用双蒸水清洗载玻片三次，每次一分钟。从载玻片上取下水滴。向载玻片中加入10至20微升抗荧光淬灭剂，并用显微镜盖玻片密封。

Research

JoVE Journal

Cancer Research

Optimizing Multiplex Immunohistochemistry (mIHC) Staining for Lung Cancer Tissue Samples to Address Autofluorescence Issues

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 ...

科研

癌症研究

Analyzing Fluorescence in Lung Cancer Tissue Samples Stained with Optimized Multiplex Immunohistochemistry (mIHC) Technique

分析使用优化的多重免疫组化 （mIHC） 技术染色的肺癌组织样品中的荧光

优化肺癌组织样本的多重免疫组化 （mIHC） 染色以解决自发荧光问题

优化肺癌组织样本的多重免疫组化（mIHC）染色以解决自发荧光问题