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The demonstration of cholinesterases by the formation of osmium blacks at the sites of Hatchett's brown

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Summary

Small amounts of Hatchett's brown (cupric ferrocyanide, Cu2Fe(CN)6·7H2O), deposited at the sites of cholinesterase activity in tissues by the procedure of Karnovsky and Roots (1964), may be enhanced by bridging to osmium through thiocarbohydrazide (TCH). Alternatively, amplification of the deposits may be attained by utilizing the cupric ferrocyanide as a catalyst to effect the oxidative coupling of 3,3′-diaminobenzidine (DAB). The resulting intensely colored osmiophilic polymer is more visible and after osmication, is electron opaque. With these procedures, the light microscope preparations are more permanent than with the Gomori modification of the Koelle and Friedenwald procedure.

One of the major advantages of this method is that thick serial plastic sections (1–2 μm) may be taken and readily studied by light microscopy until the required enzymatically stained areas appear. Thus the selection of areas for ultrathin sectioning is facilitated; enzymatically stained structures usually difficult to locate, such as intraepithelial nerve endings, are readily found.

The intensification procedures permit shorter incubation times at lower temperatures. This results in diminished tendency of the original amorphous, gel-like Hatchett's brown deposits to coalesce into relatively large cubic crystals.

Immediate rinsing of the tissues after the formation of Hatchett's brown with a 0.1 M, pH 7.2, tris (hydroxymethyl)aminomethane (TRIS) buffer prior to intensification with either TCH or DAB was found effective in eliminating some of the smaller background deposits after the longer incubations.

Studies in developing mice with an hereditary sensory neuropathy showed severe depletion of acetylcholinesterase activity concomitant with the loss of sensory endings from the rugae of hard palate, confirming the peripheral nature of the neuropathy.

High levels of acetylcholinesterase found associated with afferent trigeminal components, especially sensory endings containing clear vesicles, suggest that they may be cholinoceptive.

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Authors and Affiliations

  1. Dental Research Center, School of Dentistry, University of North Carolina, Chapel Hill, North Carolina, USA

    Jacob S. Hanker, Larry P. Thornburg, Peggy E. Yates & Horace G. Moore III

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  1. Jacob S. Hanker
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  2. Larry P. Thornburg
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  3. Peggy E. Yates
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  4. Horace G. Moore III
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Additional information

This investigation was supported by NIH research grant numbers DE 02668 and DE 00288 from the National Institute of Dental Research and by NIH grant number RR 05333 from the Division of Research Facilities and Resources.

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Hanker, J.S., Thornburg, L.P., Yates, P.E. et al. The demonstration of cholinesterases by the formation of osmium blacks at the sites of Hatchett's brown. Histochemie 37, 223–242 (1973). https://doi.org/10.1007/BF00304184

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