Visualisation of doxorubicin in human and animal tissues and in cell cultures by immunogold-silver staining

In previous pharmacologic studies, the native fluorescent properties of doxorubicin (DOX) have been utilised to visualise tissue and cellular drug distribution. Such distribution studies provide valuable additional information to that obtained by measuring tissue drug concentration alone. An alternative immunocytochemical method of drug localisation using a rabbit immunoadsorbed antiserum to DOX and silver-enhanced gold-labelled second antibodies has been used to achieve visualisation of DOX in normal and malignant tissues from drug-treated animals and patients, and in human tumour cell lines treated in vitro. Non-specific staining in untreated tissues or in controls stained without primary antibody was minimal. Widespread dark brown to black specific immunostaining was observed in the normal tissues of drug-treated animals and in rat sarcoma and in the mouse EMT6 mammary tumour. In human breast tumour biopsy samples obtained at surgery 1 h following a 25 mg intravenous dose of DOX, considerable variation in drug distribution was observed which appeared to be related to drug concentration. Both nuclear and membrane staining was apparent; the latter was especially noticeable in human tumour cells grown in the presence of DOX at concentrations greater than 0.92 microM. Immunolocalisation using silver enhanced gold-labelled reagents provides an additional technique to study cell and organ specific differences in drug uptake and distribution.