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Multiplex Fluorescence Immunohistochemistry Q&A (Part 1)
December 12, 2024
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Q1: I would like to conduct a multicolor experiment. Are there any requirements for my samples?
A1: You can preprocess your samples according to the standard procedures for immunohistochemistry. If you are selecting samples from historical sections or paraffin blocks for multicolor experiments, please try to choose relatively fresh samples (within a month). Do not select samples that have been stored for more than half a year, as they may have target loss or strong non-specificity, leading to poor staining results.
Here are the recommended procedures for processing paraffin-embedded tissue samples:
1. Obtain fresh tissue (the ideal thickness for sampling is 0.2cm to 0.3cm). If the tissue is too large, it should be cut into smaller pieces before fixation to prevent poor penetration of the fixative due to the thickness of the tissue, which could result in poor staining results;
2. Transfer the tissue to the fixative solution (commonly 10% neutral formalin or 4% paraformaldehyde) quickly after it is removed from the body (within 30 minutes). Fix the tissue at room temperature for 18-24 hours; do not fix for too long, as this can lead to loss of tissue antigens and affect the coloration of positive signals;
3. The temperature for wax infiltration should be controlled at around 58-60°C. The paraffin used for infiltration should be changed frequently to reduce the content of xylene in the paraffin. Xylene at high temperatures can make the tissue brittle and cause cell contraction, which can lead to tissue peeling off during subsequent staining processes;
4. The optimal thickness for sectioning is 4 micrometers. Tissue that is too thick can lead to poor dye penetration, and fluorescence interference between multiple cell layers can affect imaging observation. Adhesive slides specifically designed for immunohistochemistry should be used, and no adhesive should be added during the water bath slide-collecting process. After collecting the slides, place them vertically on absorbent paper to remove moisture, gently tap to remove water droplets, and do not wipe the slides with paper;
5. Bake the sections in an oven at 45°C to 65°C for about 30-60 minutes until they are dry to prevent peeling off during subsequent staining processes. However, be aware that excessively high temperatures or prolonged baking times can affect antigen activity, as high-temperature drying conditions can accelerate the oxidation of antigens in tissue sections.
Q2: Can I use frozen sections or cell smears for multicolor experiments?
A2: Yes, multicolor experiments are not limited to the type of samples you have, but it is important to ensure that your sections or smears can withstand multiple rounds of antibody elution without the samples peeling off. Only when the samples do not peel off can you achieve a good staining effect. If you are using this type of sample, you will need to prepare an additional bottle of antibody elution solution (abs994) to replace the heat repair step in the multicolor experiment. Use a gentle elution method to avoid high temperature and pressure that could damage the sections. It is important to note that you still need to control the thickness of the sections to ensure they are as thin as a single layer of cells. If they are too thick, overlapping between multiple cell layers can occur, leading to interference in the fluorescence signal. (PS: Paraffin sections are the most suitable sample type for multicolor experiments!)
Q3：How should I choose antibodies for multicolor experiments?
A3: One of the most prominent advantages of multicolor technology is that it does not have any special restrictions on the species of the primary antibodies. You can choose them based on the species of your sample. Our kit itself comes with secondary antibodies (mouse/rabbit universal secondary antibodies or anti-rabbit secondary antibodies). When selecting the source of your primary antibodies, try to choose those from rabbits or mice. If you really cannot find primary antibodies from mice or rabbits, that's also fine. You just need to prepare an additional HRP-conjugated secondary antibody that is compatible with the source of the primary antibody and can be used for immunohistochemistry (IHC). Additionally, your primary antibodies should be applicable for IHC, and it's best to choose monoclonal antibodies, as polyclonal antibodies often produce non-specific results.
Q4: I've noticed that your multicolor kit also uses fluorescent dyes. Do I need to take extra precautions to avoid light during the procedure?
A4: No, you don't need to. You can perform the procedure under normal fluorescent lighting conditions. Our dyes have high fluorescence intensity and are not that easily quenched. However, when it comes to incubation or storage, we still recommend using a light-protected humid box.
Q5: I have finished staining my multicolor slides today, but I cannot scan and observe them immediately. How should I store them?
A5: Stained multicolor slides can be stored for an extended period in the dark at 4°C, but we recommend completing imaging within two weeks after staining. If you need to store them for a long time, we suggest sealing the edges of the cover slip with clear nail polish. If the slides are stored for too long or improperly, the mounting medium may dry out, leading to strong non-specific signals when imaging. In such cases, you can use PBS to wash off the cover slip, re-mount it, and then observe, but the imaging quality will still be compromised, so we advise you to complete imaging as soon as possible. (PS: Although fluorescent dyes are not easily quenched, they can still be quenched if excited repeatedly during multiple imaging sessions. The impact may not be significant after 2-3 times, but there is always a possibility of random effects.)
Q6: How long does it generally take to complete the entire multicolor experiment? Is it possible to do it in one day?
A6: If you have a small number of markers (2-3 markers) and you are a "night owl" willing to stay up late for the experiment, then it is possible to finish it in one day, because our multicolor dyes have signal amplification capabilities, and the secondary antibodies in our kit are polymeric HRP-conjugated, which can also amplify the signal. Therefore, the primary antibody incubation that originally required overnight at 4°C can be completed in 1 hour at room temperature or in an hour at 37°C in an incubator. So, it is possible to finish the experiment in a full day. However, if you do not want to push yourself so hard, of course, you can choose to incubate the primary antibody overnight at 4°C and continue with the secondary antibody incubation and other subsequent steps the next day, just like when you perform IHC experiments. When you have more markers, you can reasonably arrange some antibodies to incubate at room temperature for 1 hour and some overnight at 4°C. But do not drag out the "battle line" too long; it is best to complete the experiment in as short a time as possible, after all, delays can lead to changes.
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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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