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Ultrastructural localization of phenothiazines and tetracycline: a new histochemical approach

SF Hoff and AJ MacInnis

To provide high resolution information on the subcellular localization of the phenothiazines and tetracycline, we have developed a new histochemical method that circumvents the difficulties inherent in classical electron microscopic tissue preparatory procedures. Specific and reliable localizations of these drugs were accomplished by their rapid precipitation with phosphotungstic acid (PTA) at pH 7. A cell suspension of Ehrlich ascites carcinoma cells was incubated with a given drug (2.5 x 10(-4) M) and then briefly cross-linked with 1% glutaraldehyde at 4 degrees C. After washing, the cells were exposed to 2% PTA (pH 7) to precipitate the drug at its binding sites. Then the samples were rapidly dehydrates in 80% ethylene glycol (4 degrees C) and embedded in the polyester, Vestopal W. This protocol provides a low denaturation, low extraction approach to tissue preparation. Control samples (without drug) demonstrated an amorphous distribution of PTA throughout the cell and no specific dense precipitates. Those cells treated with the phenothiazines (chlorpromazine or fluphenazine) or tetracycline demonstrated very discrete (4-8 nm), electron-dense drug- PTA reaction products associated with different nuclear components as well as several cytoplasmic organelles. These subcellular localizations verify the binding sites reported by the biochemical literature. In addition, several previously unresolvable binding sites are reported. The rationale and limitations of this procedure are presented. This new histochemical methodology may have broad applications in the study of drug distribution, receptors, and drug-induced pathology and toxicity that may provide new information regarding drug action and design.

Volume 31, Issue 5, pp. 613-625, 05/01/1983
Copyright © 1983 by The Histochemical Society


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