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Immunohistochemical double staining with immunogold-silver and alkaline phosphatase to identify nuclear markers of
1Department of Neurological Surgery, School of Medicine, University of California, San Francisco 94143.
Abstract:
To facilitate cell kinetics studies of brain tumors labeled with thymidine analogs, we developed a new method to identify nuclei labeled sequentially with bromodeoxyuridine (BUdR) and iododeoxyuridine (IUdR) by double staining with immunogold-silver and alkaline phosphatase. Patients received an intraoperative infusion of BUdR; excised tumor specimens were immediately labeled with IUdR in vitro, fixed with 70% alcohol, embedded in paraffin, and cut into 6 microns sections. The sections were incubated first with BR-3, a monoclonal antibody that recognizes only BUdR, and then with IU-4, a monoclonal antibody that recognizes both BUdR and IUdR; sections were counterstained with hematoxylin to identify unlabeled nuclei. Nuclei labeled only with IUdR stained red, whereas those labeled with BUdR or with both BUdR and IUdR stained black against a red background; unlabeled nuclei stained blue. This method was the most efficient differential staining technique to identify nuclei labeled only with IUdR and those labeled with BUdR among unlabeled nuclei.