Immunohistochemical detection of p53 protein overexpression in primary human osteosarcomas. Detection of p53 expression has been reported to be associated with poor prognosis in a variety of human malignancies. The aim of the current study was to utilize immunocytochemical antigen detection techniques to search for evidence of altered p53 protein overexpression in 43 primary osteosarcomas (OS). The study was carried out on formalin fixed, paraffin-wax embedded 3 to 4 microns, previously decalcified OS tissue sections. A four step biotin-streptavidin based method was employed with peroxidase conjugation as the enzymatic label. Presence of a frequent level of p53 protein expression was detected in all 43 primary osteosarcomas, suggesting a frequent p53 gene mutation. p53 protein alterations were also associated with all grades of cell microenvironment heterogeneity in the observed OSs. Overexpression of the p53 protein was detected in 31/43 (72%) primary OS cases. Formalin fixed and paraffin-wax embedded human breast carcinoma tissue sections were employed as positive control tissue. p53 protein absence was demonstrated in normal postnatal thymus, serving as the negative control tissue. Our results lead us to the following conclusions: a) the altered p53 gene product is detectable employing the chosen mouse anti-human MoAB in decalcified, formalin fixed, paraffin-wax embedded, routine tissue sections of OSs and breast carcinomas which provides an opportunity for numerous retrospective studies and comparison with additional immunodiagnostic indicators; b) primary OSs with similar cell differentiation may be genetically heterogeneous; c) p53 gene or protein alterations represent early, immunodiagnostic markers of a malignant immunophenotype (IP) in various human neoplasms, including OSs; and d) immunomorphological techniques and in situ hybridization should be employed as methods to collect data for computerized, quantitative image analysis (IA) of the cellular accumulation and localization of the altered p53 protein.