Anti-DNA polymerase alpha and Ki-67 are monoclonal antibodies that recognize nuclear antigens expressed in proliferating cells. In this study, we evaluated various methods of embedding and fixing brain tumor specimens to optimize staining with these antibodies. In fresh frozen sections, postfixation with 4% paraformaldehyde, 100% methanol, 95% ethanol and 10% buffered formalin were tested; also tested were prefixation with 4% paraformaldehyde followed by freezing and fixation with 100% methanol, 95% ethanol, or 10% buffered formalin followed by embedding in paraffin. For both antibodies, postfixation of fresh frozen sections with 4% paraformaldehyde at 4 C gave the most intense staining and lowest background activity while preserving histological features. This technique can be used in routine clinical practice to predict the growth potential of tumors.