Has anyone tried combining Immunohistochemistry using alkaline phosphatase technique and Immunofluorescence in the same sample slide?
Hi Lorena,
It is true that all the steps upto and including incubation with the primary antibody are the same, it all changes after that. The IHC relies on the physical precipitation of a chromagen and IF doesn't (unless using some sort of amplification such as Tyramide). Either way the problem is that you mount IF with aqueous media and the IHC goes through the dehydration and xylene and cover slipped with something like permount. I don't think IF is compatible with the dehydration and I doubt that IHC is works with aqueous mounting. I'm sure some one has tried though, so keep looking or just try...
Nate
Also after IHC on slide already there will be huge non-specific background for your IF. Second, there is antigen retrieval required for IHC and for IF, antigen retrieval can be skipped in many cases. So combining both IHC and IF is complicated. It is better you take two consecutive sections, let one go for IHC and second go for IF, than merge the images if you can, I am not sure that too.
Yes it is possible but you might meet some problems. Substrates might be fluorescent by itself, some form precipitates that masks, some can be dehydrated, others not. It depends also on location of the signals, if they colocalize in the same organel, I would not combine these as IF-IHC signals. But I have been doing it with success. There are fluorophores that survive dehydration and xylene, like NorthernLights (https://www.rndsystems.com/products/northernlights-fluorescent-secondary-antibodies-streptavidin-conjugates) and Cy-dyes (https://www.jacksonimmuno.com/technical/products/conjugate-selection/cyanine). Good luck!
Olav
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