Detection of Oxidized Macrophage Migration Inhibitory Factor (oxMIF) in Tissue Samples Using Immunohistochemistry...

Immunohistochemistry (IHC) staining is a technique that exploits the specificity of antibodies to detect antigens (proteins) in tissue samples. This usually involves a tissue fixation step to prevent elution, degradation, or modification of the protein of interest. However, the fixation step denatures the protein, altering the characteristic structure. This is particularly problematic when detecting oxidized macrophage migration inhibitory factor (oxMIF), the pathogenic isoform of macrophage migration inhibitory factor (MIF). In the reduced state of MIF, the oxMIF antibody epitopes are hidden within the central pore of the trimeric structure and are thus inaccessible to antibodies. OxMIF is formed through an oxidative post-translational modification of MIF in the pro-oxidative environment of inflammatory processes, as well as in tumors. This oxidation leads to a change in the native conformation of MIF, exposing these epitopes and allowing the detection of oxMIF with anti-oxMIF antibodies. Therefore, sample preparation must not artificially induce structural changes to MIF to enable specific staining of oxMIF. Here, we describe a method for detecting oxMIF in cancer tissue using IHC with oxMIF-specific antibodies that exclude cross-reactivity with reduced MIF.