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Journal of Histochemistry and Cytochemistry, Vol. 46, 11-18, Copyright © 1998 by The Histochemical Society, Inc.

ARTICLE

Freeze-fracture Cytochemistry: A New Method Combining Immunocytochemistry and Enzyme Cytochemistry on Replicas

Toshihiro Takizawaa, Takuma Saitoa, and John M. Robinsonb
a Department of Anatomy, Jichi Medical School, Tochigi, Japan
b Department of Cell Biology, Neurobiology, and Anatomy, Ohio State University, Columbus, OH

Correspondence to: Toshihiro Takizawa, Dept. of Anatomy, Jichi Medical School, 3311 Yakushiji, Minamikawachi-machi, Tochigi 329-04, Japan.

We describe a new freeze-fracture cytochemical technique consisting of combined immunocytochemistry and enzyme cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphatase activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes. (J Histochem Cytochem 46:11-17, 1998)

Key Words: freeze-fracture electron, microscopy, freeze replica, octyl-glucoside, immunocytochemistry, enzyme cytochemistry, HLA class I, CD16, CD62L, alkaline phosphatase, neutrophils


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