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New methods for the demonstration of lysosomal hydrolases by the formation of osmium blacks

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Methods are described for the direct cytochemical demonstration of the enzymes nonspecific esterase and acid phosphatase based on synthetic substrates which initially deposit Hatchett's brown (cupric ferrocyanide, Cu2Fe(CN)6·7 H2O) at their subcellular sites. The small amounts of Hatchett's brown deposited as a result of the enzyme's activity may be intensified by bridging to osmium through thiocarbohydrazide. Alternatively, even greater amplification of the sites of activity may be attained by utilizing the Hatchett's brown as a catalyst to effect the oxidative coupling of 3,3′-diaminobenzidine resulting in the formation of an osmiophilic indamine-type polymer.

One of the major advantages of this new approach is that it permits the study of acid hydrolase localization without lead in the incubation medium. Studies were performed with these methods having identical incubation media except for synthetic substrate in many different cell types and tissues. They verify a frequent nonlysosomal localization for acid phosphatase and the heterogeneity of lysosomes and lysosomal populations with respect to hydrolase content.

These methods give information obtained by direct cytochemical observation an advantage not previously held, in comparison with information from cell-fractionation cytochemical or biochemical studies. Initial studies with these methods on many tissues reinforce previous suggestions of the involvement of acid hydrolases in extralysosomal sites in subcellulur anabolic processes.

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Authors and Affiliations

  1. University of North Carolina, Chapel Hill, North Carolina, USA

    Jacob S. Hanker, Peggy E. Yates, Dorothy H. Clapp & Winston A. Anderson

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  1. Jacob S. Hanker
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  2. Peggy E. Yates
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  4. Winston A. Anderson
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Additional information

Supported by U.S.P.H.S. Grant DE-02668.

Dr. Anderson's work was performed at the Department of Anatomy of the University of Chicago and was supported by Research Grant No. M 71-077C from the Population Council.

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Hanker, J.S., Yates, P.E., Clapp, D.H. et al. New methods for the demonstration of lysosomal hydrolases by the formation of osmium blacks. Histochemie 30, 201–214 (1972). https://doi.org/10.1007/BF00277592

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