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Multicolor Fluorescent Techniques: Immunohistochemistry vs. Flow Cytometry
In the field of biomedical research, multicolor fluorescence immunohistochemistry (mIHC) and multicolor flow cytometry (FCM) are two important techniques that play a crucial role in the detection and analysis of biological samples. Although both involve fluorescence labeling and multicolor analysis, they have distinct principles, sample requirements, applications, and advantages. This article will provide a detailed comparison of these two techniques.
Principles and Characteristics
Multicolor fluorescence immunohistochemistry (mIHC), also known as tyramide signal amplification (TSA) technology, is a multiplex sequential immunostaining technique based on tyramide signal amplification. This technique utilizes horseradish peroxidase (HRP) to densely label target proteins or nucleic acids in situ, allowing the simultaneous detection of multiple targets within a single cell or tissue sample. The advantage of mIHC lies in its dual capabilities for in situ visualization and quantitative analysis, enabling comprehensive studies of cellular composition, function, and intercellular interactions.
Multicolor flow cytometry, also known as flow cytometry (FCM), is a novel high-tech cell analysis technique that integrates laser technology, photoelectric measurement technology, computer technology, fluid mechanics, as well as cell immunofluorescence chemistry and monoclonal antibody technology. FCM analyzes particles by measuring the intensity and color of fluorescence emitted from labeled cells upon laser excitation, as well as the intensity of scattered light. This technique can perform multiparameter analysis, including cell shape and size, cell cycle, intracellular cytokines, bacterial surface antigens, and cellular DNA content.
Sample Requirements
Multicolor Fluorescence Immunohistochemistry (mIHC) Sample Requirements:
1. Sample Type: mIHC typically uses formalin-fixed paraffin-embedded (FFPE) blocks or slides, including large tissue samples or tissue microarrays (TMA).
2. Tissue Fixation: Tissues should be fixed in 10% neutral formalin/polyformaldehyde for 18-24 hours.
3. Section Thickness: Sections should be approximately 4 micrometers thick, mounted on adhesive slides.
4. Sample Preservation: Preferably select relatively recent samples (within one month), with a maximum of six months to avoid loss of targets or strong nonspecific staining.
5. Tissue Processing: Transfer fresh tissue to fixative immediately, and control the paraffin embedding temperature to around 58-60°C.
6. Slide Requirements: Tissue should be tightly adhered to the slide without wrinkles, and the slide should be free of damage, scratches, or contamination.
Multicolor Flow Cytometry (FCM) Sample Requirements:
1. Sample Type: FCM requires single-cell suspensions. Peripheral blood or suspension-grown cells can be directly prepared into single-cell suspensions, while adherent cells, solid tissues, or tumor tissues need to be dissociated into single-cell suspensions.
2. Cell Viability: FCM samples must be viable, especially those that have undergone long-term transport and storage. Dead cells can cause nonspecific staining with many antibodies, making cell viability testing crucial.
3. Sample Preservation: Ideally, samples should be processed and stained immediately after collection. Different types of anticoagulants have different preservation times; for example, heparin-anticoagulated blood and bone marrow can be preserved for up to 48-72 hours at room temperature.
4. Red Blood Cell Removal: Red blood cells may need to be removed before FCM analysis, using methods such as red blood cell lysis or density gradient centrifugation.
5. Cell-to-Antibody Ratio: Adjust the cell-to-antibody ratio according to different samples to achieve optimal results.
Applications and Advantages
mIHC technology has a wide range of applications in tumor microenvironment, tumor immune infiltration, cellular senescence/apoptosis/necroptosis/autophagy, and other fields. It can obtain information on various target categories, components, and expression levels in situ within tissue cells through quantitative pathological analysis, as well as spatial localization, qualitative, and quantitative information of targets and their interactions.
1. Tumor Microenvironment Research: mIHC can simultaneously acquire information on tumor markers, cellular states, immune cell phenotyping, immune regulation, and stromal cells within tissues, providing a comprehensive analysis of the tumor immune landscape. For example, in studies of tumor immune infiltration and chemokine receptor expression, mIHC can reveal in situ interactions between different cells, which is crucial for understanding the tumor microenvironment and assessing drug efficacy.
2. Tumor Immunotherapy Efficacy Assessment: mIHC demonstrates higher accuracy in evaluating the response to anti-PD-1/PD-L1 therapy. Compared to single-marker IHC, TMB, and GEP, mIHC provides more accurate prediction of anti-PD-1/PD-L1 treatment response, with a higher area under the curve (AUC).
3. Reduced Tissue Sample Consumption: mIHC can label multiple markers on a single section, reducing sample consumption. This is particularly useful for clinical rare samples or small tissue samples obtained through biopsy.
FCM technology has a wide range of applications in cell biology, molecular biology, immunology, hematology, oncology, genetics, and other fields. It can detect cell size, granularity, DNA and RNA content, protein content, specific cell antigens, cell viability, intracellular pH, cell cycle, apoptosis, and cell function.
Applications of Multicolor Flow Cytometry (FCM):
1. Tumor Cell Parameter Detection: FCM can detect proliferation markers, differentiation/apoptosis markers, and other parameters of tumor cells, which are useful for studying the pathogenesis of tumors, developing personalized treatment plans, and making prognostic judgments. For example, DNA content analysis by FCM can serve as an important marker for the presence of malignant potential or precancerous lesions.
2. Minimal Residual Disease (MRD) Monitoring: FCM has high sensitivity and specificity in MRD monitoring for malignant hematological diseases and has become an important basis for clinical efficacy observation and prognostic stratification.
3. Tumor Immune Status Assessment: FCM can precisely assess the immune status of tumor patients through TBNK analysis, aiding in the early diagnosis of malignant tumors and providing guidance for subsequent treatment.
Summary
In summary, mIHC and FCM play different roles in biomedical research. mIHC focuses on multiparameter analysis at the tissue level, emphasizing in situ visualization and quantitative analysis, and is suitable for studying cellular composition, function, and interactions. FCM, on the other hand, focuses on multiparameter analysis of individual cells and is suitable for research in cell biology and immunology. Understanding the differences between these two techniques can help researchers choose the appropriate technical platform based on their research needs.
Recommended Products:
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Absin 7-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody) |
20T/100T |
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Absin 7-Color IHC Kit(Anti-Rabbit Secondary Antibody) |
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Absin 7-Color IHC Kit (plus) (Anti-Rabbit Secondary Antibody) |
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Absin 6-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody) |
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Absin 6-Color IHC Kit (plus) (Anti-Rabbit and Mouse Secondary Antibody) |
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Absin 6-Color IHC Kit (Anti-Rabbit Secondary Antibody) |
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Absin 6-Color mlHC Kit(plus) (Anti-Rabbit Secondary Antibody) |
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Absin 5-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody) |
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Absin 5-Color IHC Kit (Anti-Rabbit Secondary Antibody) |
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Absin 4-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody) |
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Absin 4-Color IHC Kit(Anti-Rabbit Secondary Antibody) |
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Antibody eluent (for mIHC) |
30mL |
Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.
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Absin Bioscience Inc. |
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April 23, 2025
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