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.
Shibuya
M
M
Department of Neurological Surgery, School of Medicine, University of California, San Francisco 94143.
Ito
S
S
Davis
R L
RL
Hoshino
T
T
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