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Journal of Electron Microscopy 46(1): 85-91 (1997)
© 1997 Oxford University Press

New fracture-labelling method: alkaline phosphatase in unstimulated human neutrophils

Toshihiro Takizawa* and Takuma Saito

Department of Anatomy, Jichi Medical School, 3311 Yakushiji, Minamikawachi-machi, Kawachi-gun Tochigi 329-04, Japan

*To whom correspondence should be addressed

We show a new freeze-fracture enzyme cytochemistry technique, i.e. fracture-labelling by enzyme cytochemistry. Freeze-fracture replication is carried out first and subsequently enzyme molecules in the split-membrane halves of cellular membranes are labelled with enzyme cytochemical markers. A replica-digestion treatment before the cytochemical reactions is a key step in this method. Triton X-100, saponin, and ultrasonication provided adequate cleaning of the replicas with good preservation of enzyme activity. Enzyme cytochemical fracture-labelling was applied to the study of alkaline phosphatase (ALPase)-positive granules in unstimulated human neutrophils. ALPase activity was found primarily on the exoplasmic membrane halves of intracellular small granules. Using X-ray microanalysis, we confirmed that the electron-dense deposit on replicas was the reaction product demonstrating ALPase activity. The results obtained by this method should provide unique information for the understanding of structure and functionof biological membranes.

Keywords     freeze-fracture cytochemistry,, fracture-labelling,, enzyme cytochemistry,, alkaline phosphatase,, human neutrophils

Received     24 July 1996, accepted 3 September 1996


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 J. Histochem. Cytochem.Home page
T. Takizawa, T. Saito, and J. M. Robinson
Freeze-fracture Cytochemistry: A New Method Combining Immunocytochemistry and Enzyme Cytochemistry on Replicas
J. Histochem. Cytochem., January 1, 1998; 46(1): 11 - 18.
[Abstract] [Full Text]


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