The impact of crosslinking and non-crosslinking fixatives on antigen retrieval and immunohistochemistry

Pre-analytical factors can greatly influence the outcome of molecular analyses in medical diagnostics and research. This also applies to in situ staining techniques such as immunohistochemistry (IHC), where different types of tissue fixation methods lead to different modifications of proteins and thus can affect differently the detection by antibodies. For formalin-fixed paraffin-embedded (FFPE) tissue, antigen retrieval is applied in order to reverse the negative effects of formalin and re-establish immunoreactivity. Most antibodies and protocols used in IHC are optimized for FFPE tissue, but not for paraffin-embedded tissue treated with other fixatives such as non-crosslinking fixatives. We report results from systematic studies on distinct pre-analytical conditions in IHC, immunofluorescence and electron microscopy. Parameters investigated are the impact of crosslinking and non-crosslinking fixatives (comparing formalin and PAXgene Tissue fixation) on whole tissue, subcellular structures and organelles, as well as on ultrastructure. The results generated show that minor changes in antigen retrieval conditions may have a major impact on IHC results and that protocols optimized for crosslinking fixatives may not be used for other fixatives without re-validation. Key antigen retrieval parameters such as buffers with different pH and duration of microwave treatment must be tested systematically for each antibody and fixation protocol.