Fixation of tissue specimens can be performed in various ways. The most commonly used method of fixation employs the use of an aldehyde reagent. As it relates to IHC, fixation 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 the best type of fixative to use.
Aldehydes, such as 10% neutral buffered formalin (NBF), as well as various percentages of paraformaldehyde and glutaraldehyde, are the most commonly used reagents for tissue fixation. The length of time a tissue stays in an aldehyde fixative directly affects the quality of IHC staining. The longer the fixation, the more masking that can occur. As the aldehyde fixative penetrates the tissue, methylene bridges continue to form cross-linking tissue proteins, which essentially block the epitope site.
It is always best to refer to the antibody product insert sheet for suggestions on the optimal method of fixation. The aldehyde fixative 10% NBF is considered an excellent fixative for IHC staining procedures. It is good practice to standardize the length of fixation for all specimens to ensure adequate and consistent IHC staining.
