Abstract
The development of antibodies that specifically detect histidine-phosphorylated proteins is a recent achievement and allows potential roles of histidine phosphorylated proteins in pathological and physiological conditions to be characterized. Immunohistochemical analyses enable the detection of proteins in tissues and can reveal alterations to the quantity and/or localization of these proteins through comparisons of normal and diseased specimens. However, the sensitivity of phosphohistidine modifications to phosphatases, acidic pH, and elevated temperatures poses unique challenges to the detection process and requires a protocol that bypasses traditional procedures utilizing paraffin-embedding and antigen-retrieval methods. Here, we detail a method for a brief fixation by 4% (v/v) paraformaldehyde on freshly collected tissues in the presence of PhosSTOP to block phosphatase activity, followed by a float on sucrose to protect the tissue prior to freezing. Specimens are then embedded in a cryopreservation medium in molds and frozen using an isoflurane, dry ice bath to best preserve the tissue morphology and phosphohistidine signal. We validate this technique in normal mouse liver using SC44-1, a monoclonal anti-3-pHis antibody used to uncover a role for a protein histidine phosphatase as a tumor suppressor in the liver. Furthermore, we demonstrate that the antibody signal can be eliminated by preincubating SC44-1 with a peptide treated with phosphoramidate to phosphorylate histidine residues. Thus, we present an IHC protocol suitable for specific detection of 3-phosphohistidine proteins in mouse liver tissue, and suggest that this can be used as a starting point for optimization of IHC using other phosphohistidine antibodies or in other tissue types, generating information that will enhance our understanding of phosphohistidine in models of disease.