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The Ultrastructural Demonstration of Beta Galactosidase Using the Indigogenic Technique
FORT DETRICK FREDERICK MD
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The indigogenic technique is useful for histochemical demonstration of the activity of certain enzymes, including glycosidases, beta-glucuronidase, and leucine aminopeptidase. To extend the application of this technique to the level of the electron microscope, we have studied beta-galactosidase in alveolar macrophages washed from rabbit lungs. This enzyme has not previously been demonstrated at the ultrastructural level. Preliminary studies involved the relevant properties of the indigo reaction product. Subsequently we have attempted to establish optimal conditions of fixation, incubation, embedding, and staining for demonstration of the reaction product in the electron microscope. In a comparison of fixatives, glutaraldehyde provided the best combination of preservation of enzyme activity and cell detail. Incubation times of 1 to 1 12 hours provide small but relatively easily identified crystals of reaction product. A very brief exposure of the thin section to conventional heavy-metal stain brings out cell detail without losing too much contrast of reaction product. Proof of the reliability of the technique was provided by two means 1 the reaction product was never found over nuclei, and only rarely found in extracellular areas, and 2 competitive inhibition of beta- galactosidase by galactonolactone and nonspecific inhibition of the enzyme by parachloromercuribenzoate have established the specificity of this particular substrate for this enzyme.
APPROVED FOR PUBLIC RELEASE