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 Am 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.