Immunohistochemical double staining with immunogold-silver and alkaline phosphatase to identify nuclear markers of cellular proliferation.
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.
Department of Neurological Surgery, School of Medicine, University of California, San Francisco 94143.
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Shibuya
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Ito
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Davis
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Hoshino
D000469:Alkaline Phosphatase; D015415:Biomarkers / Q000032:analysis; D001932:Brain Neoplasms / Q000737:chemistry / Q000473:pathology*; D001973:Bromodeoxyuridine; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002467:Cell Nucleus / Q000502:physiology; D005909:Glioblastoma / Q000737:chemistry / Q000473:pathology*; D006801:Humans; D007065:Idoxuridine; D007150:Immunohistochemistry
D015415:Biomarkers; D000469:Alkaline Phosphatase; D001973:Bromodeoxyuridine; D007065:Idoxuridine