Hoff S F, MacInnis A J
J Histochem Cytochem. 1983 May;31(5):613-25. doi: 10.1177/31.5.6841967.
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.
为了提供关于吩噻嗪类药物和四环素亚细胞定位的高分辨率信息，我们开发了一种新的组织化学方法，该方法克服了经典电子显微镜组织制备程序中固有的困难。通过在pH 7条件下用磷钨酸（PTA）使其快速沉淀，实现了这些药物的特异性和可靠定位。将艾氏腹水癌细胞悬液与给定药物（2.5×10⁻⁴ M）孵育，然后在4℃下用1%戊二醛短暂交联。洗涤后，将细胞暴露于2% PTA（pH 7）中，使药物在其结合位点沉淀。然后将样品在80%乙二醇（4℃）中快速脱水，并包埋在聚酯Vestopal W中。该方案提供了一种低变性、低提取的组织制备方法。对照样品（无药物）显示PTA在整个细胞中呈无定形分布，没有特异性的致密沉淀。用吩噻嗪类药物（氯丙嗪或氟奋乃静）或四环素处理的细胞显示出非常离散（4 - 8纳米）的、电子致密的药物 - PTA反应产物，与不同的核成分以及几种细胞质细胞器相关。这些亚细胞定位证实了生化文献报道的结合位点。此外，还报道了几个以前无法解析的结合位点。本文介绍了该方法的原理和局限性。这种新的组织化学方法可能在药物分布、受体以及药物诱导的病理学和毒性研究中有广泛应用，可能会提供有关药物作用和设计的新信息。
