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Cytochemical localization by ferricyanide reduction of α-hydroxy acid oxidase activity in peroxisomes of rat kidney

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Conditions are described for the use of ferricyanide as an electron acceptor for the cytochemical demonstration by light and electron microscopy of mammalian L-α-hydroxy acid oxidase activity in peroxisomes of rat kidney. Enzyme activity survives brief fixation in cold formaldehyde or in Karnovsky's fixative. Cytochemical localization of α-hydroxy acid oxidase activity in cryostat sections, or in finely chopped tissue blocks, is based on a simulaneous coupling reaction, in which ferrocyanide (produced by the enzymatic reduction of ferricyanide) is captured by copper to yield an insoluble, amorphous, electron-opaque deposit of cupric ferrocyanide (Hatchett's Brown). Under cytochemical conditions, the enzyme is most active in the presence of D,L-α-hydroxy butyric acid. The staining reaction requires the presence of substrate, and is abolished by heat treatment of sections. The use of rubeanic acid (dithiooxamide) is recommended for the visualization of the copper-containing reaction product by light microscopy. The cytochemical localization obtained is specific for peroxisomes located in cells of the proximal tubule of the rat nephron. By light microscopy, renal peroxisomes can be distinguished from lysosomes and mitochondria on the basis of their size, shape, number, and intracellular distribution. At an ultrastructural level, amorphous, electronopaque cupric ferrocyanide reaction product is precisely localized to the nucleoid and peripheral portion of the matrix of the peroxisome in lightly stained areas, and throughout the organelle, where staining is more intense. Staining results with the ferricyanide method for L-α-hydroxy acid oxidase, reported herein, are compared with those obtainable with the tetrazolium technic developed by Alien and Beard for the same enzyme, and with the 3,3′-diamino-benzidine (DAB) method for catalase.

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  1. Department of Pathology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada

    T. K. Shnitka & G. G. Talibi

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  1. T. K. Shnitka
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This study was supported by grants MT-1273 and MT-1341 from the Medical Research Council of Canada.

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Shnitka, T.K., Talibi, G.G. Cytochemical localization by ferricyanide reduction of α-hydroxy acid oxidase activity in peroxisomes of rat kidney. Histochemie 27, 137–158 (1971). https://doi.org/10.1007/BF00284956

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