Hello multiplex experts,
I am looking for a multiplex solution (4 markers or more per slide) to run immunophenotyping in joint tissue. I anticipate extremely strong autofluorescence tissue background, so fluorescent multiplex like Opal (TSA) might not be an optimal solution. Can you please advise a chromogenic kit for 4-plex for manual staining? Any comments on IHC in joint tissues will be very helpful.
Thanks,
Elena
I don’t think there is a good chromogenic 4-color system. That may explain why there are not many answers posted here...
I am not familiar with joint tissue but autofluorescence may be mainly in the green spectrum? You still can get 4 dyes that avoid the FITC/Alexa488 channel, e.g., Alexa405, Pacific Orange, Cy3, and Alexa647.
Another option is to deal with the autofluorescence by using reagents such as TrueBlack, TrueView etc. which are most likely based on some of these chemical reactions described in these publications: DOI: 10.1177/002215549904700601; DOI: 10.1016/s0165-0270(98)00066-1; DOI: 10.1007/BF00501396. I have used TrueView and it really reduces autofluorescence (and some of the real signal). Unfortunately, it seems that many of these methods to reduce autofluorescence are not compatible with tyramides conjugated to fluorescent dyes. TrueView, at least in my hands, seems to completely wipe out the tyramide signals in a multiplex stain but leaves the regular fluorescent-conjugated secondary antibodies be.
Subtracting background signal via computational methods is another option.
Actually, I stand corrected: Vector labs advertises a multi-color chromogenic system (https://vectorlabs.com/guides/multiple-antigen-labeling-guide). The question is how flexible it is and how easy it is to differentiate 4 chromogens.
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