I am looking to seek suggestions on how to pre-complex primary and conventional secondary or primary and conjugated secondary to use for IF on FFPE sections. Any tips, protocols, suggestions or ideas will be more than helpful.
This is not possible. The primary and secondary will happily bind together in the buffer you are using before the primary can interact with the antigen of interest on the tissue of interest.
You can use multiple primaries together; ex mouse and rabbit, and then use multiple secondaries together, ex. secondary anti-rabbit and anti-mouse, but you cannot use an anti-rabbit primary combined with a secondary anti-rabbit at the same time (or mouse etc).
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Not the same, but you can bind a fluorophor directly to a primary antibody with the help of commercially available labeling kits.
Hi Brent! I have used the precomplexing method successfully on OCT sections but it does not work on the FFPE sections. Hence, the curiosity. Thank you for your suggestion, I really appreciate it.
Hi Martin! I have used conjugated primary antibodies and they work well on OCT sections but not on the FFPE sections. Also, to build a panel of 50 targets, conjugating all will be a tough task vs conjugating secondary and precomplexing with the primary Ab and making that work on the sections. The precomplexing works well on the OCT sections but not FFPE.
Cool, I've never heard of this precomplexing method before. Do you have a reference I can view?
Many times with paraffin or plastic sections, I've had to include an antigen retrieval procedure prior to immunolabeling. This was not needed for cryosections using the same primaries. Have you tried an antigen retrieval step for the FFPE sections?
Hi Brent, Sure you can look up the method in:
https://www.cell.com/cell-reports-methods/pdf/S2667-2375(21)00006-0.pdf
I do perform the antigen retrieval step specific to the primary I use, even before I conjugate the Ab I IF validate it, so the whole IF protocol remains the same pretty much the same (and in principal should work ) just the primary Ab is replaced with the conjugated Ab and I do not see a staining at all. Hence, I am stuck.
Very interesting problem. If you find an answer I'd appreciate a heads up about what you find that works. Good luck (that's all I can offer).
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