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

Deciphering Tumor Microenvironment Using mIHC in Lung Squamous Carcinoma

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).

Author Spotlight: Enhancing Multicolor Fluorescence Localization in Lung Carcinoma Sample

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

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.

Otimizando a coloração por imunohistoquímica multiplex (mIHC) para amostras de tecido de câncer de pulmão para resolver problemas de autofluorescência

Para começar, obtenha fórmula previamente preparada e lâmina de amostra de tecido de câncer de pulmão fixa e embebida em parafina. Incube a 65 graus Celsius por cinco minutos. Realizar a desidratação imergindo a lâmina em xileno, seguido de soluções de etanol de concentrações decrescentes.Em seguida, mergulhe as lâminas em água esterilizada três vezes por um minuto cada. Para recuperação de antígeno, coloque a lâmina em uma caixa de coloração e reparo. Preenchê-lo com solução de trabalho de tampão de reparo de antígeno imunohistoquímico de pH seis ou nove.Cubra a caixa com uma tampa e aqueça-a no micro-ondas por oito minutos a 100% de potência. Em seguida, resfriar a lâmina até a temperatura ambiente. Cubra o tecido com uma solução de bloqueio e, em seguida, incube em uma câmara umidificada à temperatura ambiente por 10 minutos.Depois de remover a solução de bloqueio, cubra a lâmina com a solução de trabalho de anticorpos primários e coloque-a em uma câmara umedecida. Em seguida, incube esta câmara por uma hora a 37 graus Celsius. Em seguida, lave a lâmina com a solução de trabalho do tampão de lavagem por dois minutos.Adicione a peroxidase de rábano de polímero diretamente na lâmina e incube-a por 15 minutos à temperatura ambiente. Depois de lavar a lâmina, adicione a solução de trabalho de fluoróforo diretamente sobre a lâmina e incube-a por 10 minutos à temperatura ambiente. Depois que a lâmina for lavada, dê tratamento de micro-ondas como demonstrado anteriormente.Quando a lâmina for resfriada à temperatura ambiente, lave-a três vezes com água destilada dupla por um minuto cada. Em seguida, mergulhe a lâmina na solução de trabalho do tampão de lavagem por dois minutos e realize a incubação de anticorpos, peroxidase de raiz forte e adição de fluoróforo, conforme demonstrado. Para a coloração nuclear celular, cobrir a lâmina com solução de trabalho DAPI e incubá-la em uma câmara umidificada por cinco minutos à temperatura ambiente.Em seguida, lave as lâminas com água bidestilada três vezes, um minuto cada. Retire as gotículas de água do escorregador. Adicione de 10 a 20 microlitros de quencher antifluorescência à lâmina e sela-a com um vidro de cobertura de microscópio.

Research

JoVE Journal

Cancer Research

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

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

Pesquisa do câncer

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

Análise de Fluorescência em Amostras de Tecido de Câncer de Pulmão Coradas com Técnica de Imunohistoquímica Multiplex Otimizada (mIHC)