Thanks for your question about our protocol for immunohistochemistry (IHC) of formalin-fixed, paraffin-embedded (FFPE) tissue. Antigen retrieval is a critical point. Although formalin-based fixatives yield great preservation of morphology and tissue architecture, formalin fixation reduces the sensitivity of the IHC technique. Formaldehyde covalently binds to tissue protein and acts to crosslink adjoining proteins to form large accumulations of protein. The cross-linking of proteins to the antigen is supposed to “mask” the epitope and thus interfere with the binding of the antibody. So, antigen retrieval, such as heating (HIER) or enzyme processing, is necessary for FFPE tissue.
Although the mechanisms by which HIER acts are unknown, many pathologists have thought it turns back the formaldehyde-mediated chemical modifications of the antigen. Since the initial description of HIER, a wide range of buffered solutions (even “universal buffers”) have been developed. At present, HIER solutions can be divided two categories based on pH and buffer compositions, as low pH citrate buffer VS high pH EDTA buffer.
The classical theory is the knowledge that the heated efficiency of the citrate buffer-mediated HIER process breaks the cross-links that bind surrounding proteins to the antigen and open the epitope. On the other hand, EDTA buffer-mediated HIER is believed to work by removing bound calcium ions from the sites of cross-links. In this second theory, the chelation of calcium ions bound to proteins during fixation could be a critical step in HIER. Interestingly, EDTA buffers are particularly effective on over-fixed specimens and for the recovery of hard-to-detect antigens.
In our experience in long-fixed brain samples, high-pH EDTA-based solutions work well for some antigens (CD3, CD4, CD8, PD-1, PD-L1, LYVE-1, Prox1, D2-40) that are difficult to retrieve with citrate. Additionally, most phospho-tyrosine-specific antibodies appear to require the EDTA buffer (reference: Cell Signal Technology brochure). In our study of the dural lymphatic system, the EDTA buffer showed very specific signal with less background than citrate, so we recommend EDTA for LYVE-1 IHC in FFPE tissue.
2019-09-17 16:38:15