Fixation can be performed in various ways. The most commonly used fixation method is with an aldehyde reagent. Fixation for IHC can cause masking or blocking of the epitope/antigen of interest. If the tissue has a low abundance of epitopes of interest, one must consider what type of fixative to use.
Aldehydes, such as 10% neutral buffered formalin (NBF), various percentages of paraformaldehyde, and glutaraldehyde are the universal reagents used in tissue fixation. The length of time that a tissue stays in an aldehyde fixative is crucial in IHC staining. The longer the fixation, the more masking can occur as methylene bridges are forming, cross-linking tissue proteins during aldehyde fixation. Inadequate fixation also impairs the tissue and does not allow binding sites to be made available even after the harshest antigen retrieval methods.
It is always best to refer to the antibody data sheet for suggestions on the optimal type of fixative method. Of all the aldehydes, 10% NBF is considered an excellent fixative for IHC procedures. It is good practice to standardize fixation to 24 hours for all specimens to ensure adequate and consistent IHC staining.
