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

Расшифровка опухолевого микроокружения с помощью мИГХ при плоскоклеточной карциноме легкого

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).

В центре внимания автора: Усиление многоцветной флуоресцентной локализации в образце карциномы легкого

Мультиплексное иммуногистохимическое окрашивание тканей рака легкого, залитых парафином

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.

Оптимизация мультиплексного иммуногистохимического (мИГХ) окрашивания образцов тканей рака легкого для решения проблем аутофлуоресценции

Для начала возьмите заранее приготовленную формулу и фиксированный и залитый парафином образец ткани рака легких. Выдерживайте его при температуре 65 градусов Цельсия в течение пяти минут. Выполните обезвоживание путем погружения предметного стекла в ксилол, а затем в растворы этанола с уменьшающейся концентрацией.Затем погрузите горки в стерилизованную воду три раза по одной минуте каждый. Для извлечения антигена поместите предметное стекло в коробку для окрашивания и ремонта. Заполните его иммуногистохимическим буфером восстановления антигена рабочим раствором pH шесть или девять.Накройте коробку крышкой, и разогрейте ее в микроволновой печи в течение восьми минут на 100% мощности. Затем охладите горку до комнатной температуры. Накройте ткань блокирующим раствором, а затем выдержите во влажной камере при комнатной температуре в течение 10 минут.После удаления блокирующего раствора накройте предметное стекло рабочим раствором первичного антитела, и поместите его во увлажненную камеру. Затем инкубируйте эту камеру в течение одного часа при температуре 37 градусов Цельсия. Далее промойте предметное стекло промывочным буферным рабочим раствором в течение двух минут.Добавьте полимерную пероксидазу хрена непосредственно по капле на предметное стекло и инкубируйте его в течение 15 минут при комнатной температуре. После промывки предметного стекла добавьте рабочий раствор флуорофора непосредственно по капле на предметное стекло и выдержите его в течение 10 минут при комнатной температуре. После того, как предметное стекло будет вымыто, проведите лечение в микроволновой печи, как было показано ранее.Когда предметное стекло остынет до комнатной температуры, промойте его три раза двойной дистиллированной водой по одной минуте каждый. Затем опустите предметное стекло в рабочий раствор промывочного буфера на две минуты и выполните инкубацию антител, пероксидазу хрена и добавление флуорофора, как показано на рисунке. Для окрашивания ядра клетки накройте предметное стекло рабочим раствором DAPI и выдержите его во влажной камере в течение пяти минут при комнатной температуре.Затем промойте горки дважды дистиллированной водой три раза, по одной минуте каждый. Удалите капли воды с горки. Добавьте от 10 до 20 микролитров антифлуоресцентного гасителя на предметное стекло и закройте его покровным стеклом микроскопа.

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 ...

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

Анализ флуоресценции в образцах тканей рака легкого, окрашенных с помощью оптимизированного метода мультиплексной иммуногистохимии (мИГХ)