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Multiplex Fluorescence Immunohistochemistry Q&A (Part 4)
December 17, 2024
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Introduction
Multicolor fluorescence immunohistochemistry technology, based on tyramide signal amplification (TSA), is a class of enzymatic detection methods that utilize horseradish peroxidase (HRP) to perform high-density in situ labeling of target proteins or nucleic acids.
In the presence of hydrogen peroxide, the dye-labeled substrate tyramide molecules are transformed into a short-lived active intermediate state by HRP that is fixed by antibodies or probes, and then the activated intermediate molecules rapidly and stably covalently bind to the electron-rich regions (tyrosine residues) of adjacent protein molecules.
Figure 1: Horseradish Peroxidase (HRP) Reaction of Tyramide
Why Choose Multicolor Technology? Advantages of Multicolor Technology
1. Simultaneous labeling of multiple proteins (up to 9 markers with 10 colors);
2. No longer limited by the source of primary antibodies (same species multi-markers);
3. Connection of horseradish peroxidase-conjugated same-species secondary antibodies;
4. Ultra-strong fluorescence with anti-quenching properties.
This issue mainly addresses some of the customers' doubts about the instruction manual, covering all common questions in one go!!!
Figure 2: Absin mIHC Kit
Analysis of FAQs for Absin mIHC Kit Manual:
1. What is the purpose of the step "10% neutral formalin immersion for 10 minutes, sterilized water rinse for 1 minute, repeat 3 times" in the dewaxing and hydration process? We didn't have this step when we made paraffin sections before.
Answer: This is a post-fixation step, which can be omitted if the tissue is sufficiently fixed.
2. Is antigen retrieval required for every round of staining? Can it be replaced with antibody elution buffer? In which step is the antibody elution buffer used?
Answer: Antigen retrieval is performed before staining the first antibody to expose the antigenic determinants that may have been sealed by paraffin or fixatives, allowing antibodies to recognize the antigens. Antigen retrieval before staining subsequent antibodies is actually for removing the antibodies from the previous round and any unbound dyes, which is more accurately referred to as "antibody elution." This "antibody elution" can be replaced with antibody elution buffer (e.g., ABS994), especially for cell, frozen sections, or bone samples. However, the first antigen retrieval cannot be replaced by antibody elution buffer. Frozen sections can omit antigen retrieval, but if the staining effect is poor, antigen retrieval can be considered. It is recommended to use a rapid antigen retrieval solution (5×) (e.g., ABS9207) for frozen sections.
3. Is endogenous peroxidase blocking necessary? At which step should it be performed? Is it required for every round?
Answer: Endogenous peroxidase blocking is necessary. The detailed procedure is to perform it before serum blocking with a 3% hydrogen peroxide methanol solution at room temperature for 10-15 minutes, followed by three washes with PBST, and then serum blocking. Peroxidase blocking should only be done once.
4. Is antigen retrieval (referring to antibody elution) required before staining with DAPI?
Answer: In theory, it is required. However, if it is a frozen section that has undergone multiple elutions, there is a significant risk of tissue detachment. In such cases, it is better to have a less clean background rather than tissue detachment, so antigen retrieval can be omitted at this step.
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Item NO. |
Product Name |
Size |
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Absin 4-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody) |
20T/100T |
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Absin 4-Color IHC Kit(Anti-Rabbit Secondary Antibody) |
20T/100T |
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Absin 5-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody) |
20T/100T |
|
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Absin 5-Color IHC Kit (Anti-Rabbit Secondary Antibody) |
20T/100T |
|
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Absin 6-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody) |
20T/100T |
|
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Absin 6-Color IHC Kit (Anti-Rabbit Secondary Antibody) |
20T/100T |
|
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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) |
20T/100T |
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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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