Studies on reaction and binding of monoamines after fixation and processing for electron microscopy with special reference to fixation with potassium permanganate

Summary In the present paper certain properties of potassium permanganate (KMnO₄), a fixative used for electron microscopical investigations, have been studied in model test tube experiments and on tissues. Evidence was obtained that KMnO₄ reacts with different types of biogenic monoamines resulting in a formation of a precipitate. In addition, also various monoamine analogues, precursors and metabolites reacts with KMnO₄. The reaction taking place may be an oxidation-reduction-reaction in which KMnO₄ is reduced, probably mainly to manganese dioxide by hydroxyl groups of the amines and related compounds. This is corroborated by the fact that no reaction takes place between KMnO₄ and -phenylethylamine or amphetamine, two substances, which lack hydroxyl groups.Using labelled monoamines evidence was obtained that the amine partly is retained within the precipitate formed after the reaction with KMnO₄ and also in tissues fixed with KMnO₄, indicating a possibility to perform autoradiographic studies on KMnO₄ fixed tissue.Electron microscopic studies on tissues fixed under various conditions revealed that fixation with low concentrations (0.6 and 1.0%) of KMnO₄ and at high temperatures (about 20° C) leads to inferior results as to general morphology and as to the visualization of intraneuronal amine stores.Different types of permanganates were tested as fixatives. These results show that fixation with permanganates with monovalent metallic ions (K⁺, Li⁺ and Na⁺) give good results of comparable quality, whereas fixation with zinc permanganate results in seriously destroyed tissues. However, tissue fixed with calcium permanganate reveals very distinct membranes. Furthermore, evidence was obtained that fixation with high concentrations of LiMnO₄ (6 and 9%) and NaMnO₄ (6 and 9%) was more sensitive as to the demonstration of monoamines at the ultrastructural level as compared to 3% KMnO₄. Thus, with e.g. 6 and 9% LiMnO₄ small granular vesicles could be seen in slices from the caudate nucleus after incubation with -methyl-dopamine. This was not possible when using 3% KMnO₄ as a fixative.