A double staining method for histone H3 mRNA and p53 protein in oral tumors using in situ hybridization and immunohistochemistry

T. Nishikawa, PhD, Y. Ito, DDS, R. Hara, DDS, M. Wato, PhD, A. Tanaka PhD.

Department of Oral Pathology, Osaka Dental University 8-1, Kuzuhahanazono-cho, Hirakata-shi, Osaka 573-1121, Japan

AIM: Proliferative and aberrant cellular activities of human tissues were evaluated using a novel double staining technique for histone H3(with in situ hybridization and immunohistochemistry for p53 protein. METHODS: Simultaneous analyses were run on tissue sections of papi11omas, squamous cell carcinomas (SCC) and normal non-neoplastic stratified squamous epithelia from human oral specimens using formalin-fixed, paraffin-embedded material. Sections were first FITC-labeled histone H3 DNA probe and then treated with alkaline phosphatase-conjugated anti-FITC rabbit antibody. Following reaction with anti-p53 protein mouse monoclonal antibody using the labeled sterptavidin-biotin (LSAB) method, sections were stained with 3-amino-9-ethylcarbazole-HCl to detect P53 protein, and with 5-bromo-4-chloro-3-indoxy phosphate/nitro blue tetrazolium to detect histone H3 mRNA. Several methods differing only in the order of detection procedures were employed and subsequently compared. RESULTS: Using these methods, histone H3 mRNA and p53 protein were detected both within the cytoplasm and the nucleus respectively. Histone H3 mRNA and p53 protein were expressed in larger ce11 populations in the following order: SCC, papilloma, and non-neoplastic stratified squamous epithelia. CONCLUSIONS: The double staining as employed here proved defective for simultaneous evaluation of cell proliferative activity as well as the overexpression of aberrant gene on the same tissue sections.

KEY WORDS: squamous cell carcinoma, histone H3 mRNA, p53 protein, in situ hybridization, immunohistochemistry.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Diagnostic Markers.