Choosing the Right Tissue Prep for Tau IHC
The neurodegeneration-related protein tau holds molecular clues to how diseases like frontotemporal lobe dementia and Alzheimer's disease are driven and can be treated. To answer questions about the mechanism and pathology of disease, researchers often depend on detecting tau via immunohistochemistry (IHC), which uses antibodies to produce a chromogenic or fluorescent stain of a particular target protein. In the case of tau, there are hundreds of antibodies that allow for the detection of this neurological target, including antibodies specific for post-translationally modified (PTM) variants of tau, such as phospho-tau.
When performing IHC staining, a fork in the road that researchers face is the decision of using frozen or formalin-fixed paraffin-embedded (FFPE) sample preparation protocols. By carefully considering this decision upfront, researchers can save time troubleshooting and optimizing. While this is true for most proteins, it becomes especially important in the study of the protein tau.
This decision is important because IHC using frozen tissues (IHC-Fr) versus using FFPE tissues (IHC-P) differ drastically in how morphology is preserved and how antigens are detected. While it is important to preserve tissue for all IHC applications, frozen and FFPE tissue are preserved in different ways. As the name suggests, IHC-Fr requires extreme cold for tissue preservation. Tissue is embedded in OCT compound frozen prior to cryosectioning, and then fixed to the slide in an organic solvent such as acetone or methanol.IHC-P, on the other hand, uses formalin to cross-link proteins to prevent autolysis and preserve structure; tissue is then embedded in paraffin wax that must eventually be removed.
The sample preparation for IHC-P has an advantage for long-term storage at ambient temperatures, but more importantly preserves the overall morphology of the tissue better than IHC-Fr. However, FFPE tissue may obscure antigens due to chemical crosslinking, which can make it more difficult for even the best antibodies to detect targets, especially those at lower cellular abundance. For this reason, researchers often must perform special buffered washes or apply heat to retrieve antigens from FFPE tissue. IHC-Fr, while preserving less morphology, offers the advantage of detecting antigens with ease.
When it comes to IHC staining of tau, the research question should play a role in dictating which sample preparation method would be optimal. The biology of tau reveals that PTM status plays a major role in disease pathogenesis. Pathologic tau assembles into specific structures known as neurofibrillary tangles. The decision of FFPE versus frozen tissue depends on which of these aspects of tauopathy is being measured. Essentially, a researcher must determine whether they are querying tau at a molecular level or a cellular level.
A researcher who is studying tau on the molecular level may be interested in the specific PTM status of tau, asking what causes tau to become phosphorylated at a particular site, and what signaling steps lead to this dysregulated proteoform. In this case, using IHC-Fr would be an optimal sample preparation protocol for the study of tau because the priority is antigen detection rather than morphology. Though heat-induced epitope retrieval (HIER) and other antigen-retrieval methods technically make it possible to use FFPE tissue with an antigen-focused question, IHC-Fr does so with less risk. Especially in the case of phosphorylation, which can be heat-sensitive, IHC-Fr accesses tau as an antigen target much more effectively than IHC-P.
On the other hand, a question about tau at the cellular level — particularly regarding the consequences that drive neurofibrillary tangle formation and other forms of tau aggregates – would be poorly served by IHC-Fr. Morphology is not completely lost in IHC-Fr, but IHC-P outperforms it in this regard. To efficiently detect tau aggregates through IHC, FFPE will preserve the large scale morphology and preserve tissue anatomy in a way unrivaled by IHC-Fr.
Not sure which sample preparation protocol is optimal for your next tau experiment? Need help finding the right antibody to answer your question about neurodegeneration? Feel free to reach out with technical questions and troubleshooting to our human-monitored support inbox at [email protected].
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