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Multiplex Fluorescence Immunohistochemistry Q&A (Part 3)
December 16, 2024
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Q1: How can I achieve perfect multicolor results?
A1: First, you need to have good quality samples and antibodies, which is already half the battle (sample and antibody selection strategy). For samples, tissue detachment is the "number one enemy" we need to prevent. Multicolor experiments require repeated heat repair and antibody elution, and if the slides detach, it can lead to blurred imaging at best and tissue loss at worst, rendering all efforts in vain. You can simulate a few rounds of heat repair at the beginning of the experiment, without adding antibodies and other reagents, to see if there are signs of tissue detachment. If detachment is severe, you may need to prepare new sections. If there is slight detachment, you can consider purchasing antibody elution solution (abs994) to replace the heat repair process. Then, you can start the experiment. We recommend that you first explore the working conditions for antibodies and dyes before proceeding with multiple staining. This way, the success rate of multiple staining can be over 90%! For the final 10%, it's necessary to adjust the combination of markers and dyes as well as the order of staining.
Q2: Are there any recommended dye combinations and staining orders?
A2: The general principle for dye matching is "pair weak targets with strong dyes, and strong targets with weak dyes." This means that markers with weaker expression should be paired with dyes that have stronger fluorescence signals, while markers with stronger expression should be paired with dyes that have weaker signals. For Absin's TSA dyes, the shorter the wavelength, the stronger the fluorescence intensity. For example, if you need to stain PD-1, CD8, and CD3, and you have a four-color kit (TSA520/TSA570/TSA650), and through IHC you find that the sample has very little PD-1 signal and a lot of CD3 signal, with CD8 in between, then it would be reasonable to pair TSA520 with PD-1, TSA650 with CD3, and TSA570 with CD8.
Next is how to arrange the order of staining:
(1) Divide cell localization, from outside to inside: Determine the cell localization for each marker, stain membrane expressions first, and cytoplasmic or nuclear expressions later (if there are many markers, intracellular markers should be stained later, after several rounds of elution, cell permeabilization may not be necessary; if there are few markers, cell permeabilization is still required before staining intracellular markers, and multiple intracellular markers only need to be permeabilized once);
(2) Determine antigen retrieval methods, from acid to base: Base retrieval is stronger than acid retrieval, and some antibodies may show a lot of non-specific staining after base retrieval, so if they are the same cell localization, stain with acid retrieval first, then with base retrieval antibodies;
(3) Based on IHC results, from weak to strong: If the IHC results show that the marker is relatively rare and weak, it should be stained first. In theory, the earlier the marker is stained, the closer the effect will be to single-marker staining.
The above are some general rules, but different markers may still vary, and to achieve the most perfect results, you would need to try different combinations one by one (as shown in the table below, "X" represents a signal). This can be time-consuming and require a lot of reagents. You can first perform a multi-staining based on the general rules, and then compare each channel with fluorescent single-staining. If the results are significantly different, then adjust the staining order. If the single-staining results differ greatly from the IHC (showing high background or significant non-specificity), consider changing the dye combinations.
| Target | Best Signal | Recommended Dilution |
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| 1st AR | 3rd AR | 6th AR | ||
| FOXP3 | x | 1:250 | ||
| PD-1 | x | 1:250 | ||
| LAG3 | x | x | 1:250 | |
| CD4 | x | x | 1:250 | |
Q3: After I finish imaging, I want to do data analysis. Do you have any software recommendations?
A3: There are quite a few data analysis software options on the market. For free options, you can download ImageJ or QuPath, but the downside is that you'll need to figure out how to use them on your own. Our company has installed HALO and StrataQuest, with HALO being a paid software and StrataQuest coming with our TG scanner platform. In terms of functionality, analysis accuracy, and operability, they are definitely superior to open-source software, and they come with a professional technical support team to answer questions. You can choose the analysis software that suits your actual situation. If you're really at a loss, you can also reach out to Absin, as we can provide data analysis services.
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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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