Indirect Immunofluorescence Detection of Protein-Bound 4-Hydroxynonenal in Tissue Sections and Isola

4-Hydroxynonenal (4-hydroxy-2,3-trans-nonenal; 4-HNE) is the best known and thoroughly studied aldehydic product originating in biological samples during the process of lipid peroxidation (Fig. 1 ) ( 1 ). The latter is an autocatalytic, self-propagating sequence of free radical reactions, ultimately resulting in the fragmentation of the carbon atom chains of unsaturated fatty acids esterified in phospholipids of cellular membranes, which can be set into motion in conditions of severe oxidative stress within the cell ( 2 ). Many of the lipid fragments thus originated are aldehydes and other carbonyl products, provided with variable reactivity towards cellular macro molecules. 4-HNE was originally identified in vitro as a specific, dialyzable, cytotoxic product of peroxidation of microsomal phospholipids ( 3 ), but subsequent studies have consistently detected it in a number of experimental conditions, in which it has been shown to exert a variety of biological actions ( 4 ), as well as in important human diseases such as atherosclerosis, neurodegeneration, and cancer ( 5 – 7 ) Like other α,β-unsaturated aldehydes, 4-HNE is capable of binding covalently to side chains of cysteine, histidine, lysine, and other amino acids in proteins ( 8 ), thus originating new epitopes that can be detected by suitable antibodies Fig. 2 ). Here a convenient procedure is described using polyclonal antibodies (PAbs) and fluorescent revelation.   Fig. 1.  Proposed mechanism for the formation of 4-hydroxy-2,3- trans -nonenal (4-HNE) from peroxidation of a phospholipid (PL) containing linoleic acid.   Fig. 2.  The formation of new epitopes in protein following the binding of 4-HNE to representative amino acid residues.