Multiplex IHC is based on multiple rounds of incubating/detaching antibodies (+6 ABs) to assess expression of several proteins on the same sample. However, I didn´t find so far an explanation of how the detaching rounds are not affecting other structures in the sample (other proteins), but only the antibody/antigen bond (which is covalent)?
Antibodies do not bind covalently to their target epitopes. However, the fixation process produces covalent cross-linking in the tissue samples that stabilize the endogenous proteins.
Most typical IHC protocols include a heat-induced epitope retrieval step, which will disrupt the antibody-epitope binding from previous rounds of IHC. Additional blocking steps, such as the use of HRP-binding Fab fragments, are also used to prevent any background signal from previous staining rounds.
When you optimize your multiplex panel, you should investigate which epitopes are more sensative too heat deinactivation. You can put the strongest epitopes at the end of the multiplex and more sensitive ones in the beginning. This way you wont lose any signal.
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