Hi,
I routinely incubate FFPE samples with different primary antibodies to specific target proteins for IHC. The primarys are always rabbit IgG, usually monoclonal. We then use the same secondary antibody in every experiment: F(ab´)2 anti-rabbit IgG HRP. I was wondering if it possible to strip the secondary antibody only from the tissues, whilst leaving the primarys intact and still bound. Traditional stripping options like microwaving or HIER in a TP-link will naturally remove the primary also, and I need to avoid this in the experimental design.
NOTE: this is not a multiplexing experiment, so changing antibody pairs etc is not something I am interested in doing. Stripping the secondary, whilst leaving the primary, is a must. Can binding between the afformentioned antibodies be distrupted, or enzymatically targeted perhaps?
Any insights or suggestions would be great.
Thanks
To detach the secondary antibody try to use first PBS - tween tandem wash and leave overnight in the fridge.
If this does not work,
I am not sure but stripping with excess primary antibody Fc may also remove the secondaries by competitive binding and/or high-concentration-induced displacement.
I think that Ismail's suggestion of using competitive binding with free Fc is the only thing that will work. Detergents are more likely to remove the primary antibody since the avidity of secondary antibodies is generally far greater than that of primary antibodies.
Fixing with NBF after primary incubation comes to my mind, that would bind it covalently.
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