Multiplex Fluorescence Immunohistochemistry Q&A (Part 2)

Q1: Why is antigen retrieval necessary after staining each antibody? Can I only do it for the first marker and skip it for the rest?

A1: No, antigen retrieval is required after staining each antibody. Performing antigen retrieval before staining the first antibody is to expose the antigenic determinants that have been sealed by paraffin or fixatives, allowing the antibodies to recognize the antigens. Antigen retrieval before staining each subsequent antibody is aimed at removing the antibodies from the previous round of staining and any unbound dyes that remain. More accurately, this process should be called "antibody elution," because in multi-color experiments, there is no restriction on the source of the primary antibodies. If all your primary antibodies are from rabbits, then the secondary antibodies used for each primary antibody are anti-rabbit. If we do not elute the previous primary antibody, the secondary antibody will also bind to the previous round's primary antibody when staining the second antibody, resulting in large areas of non-specific staining. Therefore, antibody elution is an indispensable core step in the entire multi-color experiment.

Q2: Do I need to change the antigen retrieval solution (antibody elution solution) for each round of retrieval?

A2: Yes, you do. Firstly, our antigen retrieval solution needs to be selected based on the primary antibody to be stained in the next round. Some primary antibodies require citrate retrieval, while others may need EDTA retrieval. The antibody manufacturers often specify what retrieval solution their antibodies require. Secondly, even if all our primary antibodies are recommended to be retrieved with citrate, after each elution of a marker, the retrieval solution will contain residues of the antibodies from the previous marker. If the retrieval solution is reused, it can affect subsequent markers, leading to non-specific staining.

Q3: Can I still perform multi-color staining if my tissue has autofluorescence?

A3: Generally, yes, it is possible. First, you can directly observe the autofluorescence of your unstained slides (tissue sections without any treatment) using a fluorescence imaging device. You can try imaging each channel to see which channel or channels the autofluorescence is observed in. After determining the approximate wavelength range of the autofluorescence, we should avoid wavelengths where autofluorescence is present when selecting multi-color dyes. For example, if your autofluorescence is observed in the FITC channel and not in others, and the corresponding dye from Absin that overlaps is TSA520, you simply avoid using TSA520 when performing multi-color staining. Of course, to avoid the autofluorescence channels, we may have to give up some dyes, which will correspondingly reduce the number of colors that can be stained. Moreover, if you find that your sample shows signals in all fluorescence channels of your instrument, then this sample is not suitable for multi-color staining, and you will need to prepare a new sample.

Q4: Is there a significant difference in staining results between microwave retrieval, high-pressure retrieval, and antibody elution retrieval?

A4: The impact of retrieval methods on staining results actually depends on the primary antibody and the sample. For most paraffin samples, we recommend high-pressure heat retrieval, which is a widely recognized effective method. The Absin laboratory has also been using high-pressure heat retrieval. Of course, microwave heat retrieval can also yield good results, but the performance of different microwave ovens varies, so the working conditions cannot be completely replicated and require some exploration. Additionally, microwave heat retrieval may take a longer time, which can easily lead to the evaporation of the retrieval solution and affect the results. Antibody elution retrieval is mainly used for samples that cannot be treated with heat retrieval or are prone to detachment with heat retrieval (such as frozen sections, hard bone sections, etc.). The working conditions are milder, and the elution effect depends on the thickness of the sections, the elution temperature and time, and the type of primary antibody (elution difficulty: structural membrane proteins and cytoskeletal proteins > cytoplasmic proteins > nuclear proteins). For samples that can withstand multiple rounds of heat retrieval, we recommend using heat retrieval.

Q5: What other materials do I need to prepare for multi-color experiments besides the multi-color kit?

A5: Our kit includes TSA fluorescent dyes, dye dilution solution, secondary antibodies, DAPI, and anti-fluorescence quenching sealing agent. Apart from these, you will need to prepare other reagents on your own, following the preparation for immunohistochemistry. The most important thing is that you need to have a fluorescence imaging device.

Q6: Our lab has a fluorescence device, but I'm not sure if it can be used to observe multi-color experiments.

A6: You will need to confirm the excitation and emission wavelengths of each channel of your fluorescence device (you can consult the instrument manufacturer) and match them with the wavelengths of our fluorescent dyes (see the table below). We provide the optimal wavelengths, at which the best imaging results can be achieved. However, if the parameters differ by 30nm, it is still possible to observe. If you really cannot determine the specific instrument parameters, you can also do some approximate wavelength matching. For example, if you know your microscope can observe FITC, then the corresponding Absin multi-color dye is approximately TSA520. If you find that some channels of your instrument are not on our approximate wavelength list, such as Texas Red, you can look up some materials to confirm the excitation and emission wavelengths of that dye (Texas Red EX/EM: 570/600nm), and then match it with Absin's dye parameters, which are close to TSA620 (EX/EM: 590/620nm). Both excitation and emission wavelengths are within a 30nm range, so you can determine that this channel can observe the TSA620 dye. However, approximate dye matching may still result in the inability to observe, and confirming the instrument parameters is the most accurate method. If, after staining, you find that imaging is indeed not possible, you can also consult Absin's scanning service.

Table 1: Absin Multi-color Fluorescent Dye Wavelength Matching Chart

Dye Name	Wavelength		Similar Fluorescent Dyes
	Excitation Wavelength（nm）	Emission Wavelength（nm）
DAPI	360	461	DAPI
TSA Monochromatic Fluorescent Dye 480	430	480	Opal480
TSA Monochromatic Fluorescent Dye 520	490	520	FITC/AF488/Opal520
TSA Monochromatic Fluorescent Dye 540	520	540	AF514/Opal540
TSA Monochromatic Fluorescent Dye 570	550	570	CY3/AF555/Opal570
TSA Monochromatic Fluorescent Dye 620	590	620	AF594/Opal620
TSA Monochromatic Fluorescent Dye 650	640	660	AF610/Opal650
TSA Monochromatic Fluorescent Dye 670	650	670	APC/CY5/AF647/Opal670
TSA Monochromatic Fluorescent Dye 690	660	690	AF660/CY5.5
TSA Monochromatic Fluorescent Dye 700	682	702	AF680/CY5.5/Opal690
TSA Monochromatic Fluorescent Dye 770	740	780	Opal780

Item NO.	Product Name	Size
abs50012	Absin 4-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)	20T/100T
abs50028	Absin 4-Color IHC Kit(Anti-Rabbit Secondary Antibody)	20T/100T
abs50013	Absin 5-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)	20T/100T
abs50029	Absin 5-Color IHC Kit (Anti-Rabbit Secondary Antibody)	20T/100T
abs50014	Absin 6-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)	20T/100T
abs50030	Absin 6-Color IHC Kit (Anti-Rabbit Secondary Antibody)	20T/100T
abs50015	Absin 7-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)	20T/100T
abs50031	Absin 7-Color IHC Kit(Anti-Rabbit Secondary Antibody)	20T/100T
abs994	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.

Absin Bioscience Inc. Email: worldwide@absin.cn

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