Localization of Intracellular Lipid Hydroperoxides Using The Tetramethylbenzidine Reaction for Transmission Electron Microscopy

Histochemical reactions for lipid hydroperoxides (LHP) using indophenol, benzidine, or phenylendiamine as the electron donor have been described previously for auto-oxidized adipose (1 ) and neuronal (2 ) tissue. More recently, tetramethylbenzidine (TMB) has been proposed as another chromagen (3 ). The usefulness of the TMB reaction for ultrastructural studies of lipid peroxidation was demonstrated in retina where LPH was generated by incubation with exogenous lipoxygenase. The glutaraldehyde fixed tissue, which was reacted with TMB and then postfixed in osmium tetroxide, showed an electron-dense product (4 ). This technique allows intra- and extracellular localization, as well as a comparison of relative intensity amoung various cell types and subcellular organelles (5 ). In light-induced lipid peroxidation, discs of the outer segments, which are rich in oxidizable long-chain polyunsaturated fatty acids, stain strongly and appear as bubble-like structures (6 ). These are however, quite similar to fingerprint profiles seen acutely in outer segments and chronically in neurons, which are visualized without TMB following exogenous exposure to LHP (7 ,8 ). A possible caveat to the reported method is that peroxidized protein and carbohydrates many also react and so the TMB method has not been proven to be specific for LHP only.