Hi all,
I am trying to combine 4 different markers for my multiplexed IHC.
Their Antigen Retrieval (AR) conditions are as follows:
Marker A: pH9 microwave, 30 mins
Marker B: pH9 microwave, 15 mins
Marker C: pH6 steamer, 20 mins
Marker D: pH6 microwave, 15 mins
As you can see, two of my markers have pH9 AR conditions and the other two have pH6 conditions. The efficiency and specificity of these antibodies have already been verified by singleplex staining. After weeks of optimizing my staining sequence, I've come to realize that AR conditions for one antibody might be affecting the other. In my case, AR conditions of Marker A and B affects staining of marker C (marker D remains unaffected). I've also tried doing AR at pH6 followed by pH9 (following the staining sequence C-->D-->A-->B). Even in this case, staining of marker-A is affected by staining conditions of C and D. Thus far, I've noticed that only Marker A and C are sensitive to change in pH of the antigen retrieval process. The other two markers, B and D remain unaffected by the change in pH conditions.
Have you faced a similar problem with your experiments ? If yes, is there a way to overcome it?
Thanks in advance
Hi Teena,
I have encountered this effect as well. But in my case the effect was positive. The heattreatments seemed to increase the staining-intensity of the markers.
If I were you, I would try a combination starting with A/C or C/A.
You could also try to perform both AR methods before staining.
(pH9 microwave --> steamer pH6 --> staining A --> staining B; only possible if antibodies for A and B are different species and if you use different enzymes for development).
I think you will have to determine empirically what is the best option.
with kind regards,
Ronald
Hi Teena
In principle you could also have false positives due to an unappropriate or too strong AR conditions. My advice is to stain for max 3 antigens at once.
I sounds like you are using tyramide amplification? That is a knowm problem because some antigens get degraded after too long antigen retrieval whereas others show higher retrieval rate after longer antigen retrieval. I guess the changing of the buffers can also have effects which are unknown. You can only try too switch the order of your antibodies and see if you get better results.
Your retrieval times seem quite long too me. Usually it is enough to retrieve for 5 min to strip away the antibodies from before and 10-20 min the very first time. I would also recommend a steamer for the first step antigen retrieval or even all of them.
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