METHODS IN PATHOLOGY - P53 IMMUNOLABELING IN ARCHIVAL PARAFFIN-EMBEDDED TISSUES - OPTIMAL PROTOCOL BASED ON MICROWAVE-HEATING FOR 8 ANTIBODIES ON LUNG CARCINOMAS

The prognostic value of p53 gene mutations is dealt with by several re cent reports. However, retrospective assessment of p53 tumor status on archived samples has been prevented by p53 epitope alteration during routine fixation and embedding procedures. This study aimed at establi shing a reproducible low-cost protocol to retrieve not only N-terminal , but also midregion and C-terminal, epitopes, with special attention to possible artifacts induced by epitope retrieval procedures. Using m icrowave heating, we compared the epitope retrieval efficiency of five solutions with eight commercial antibodies on 21 lung carcinomas for which frozen tissue and samples fixed with formalin and Bouin's liquid were available. All eight epitopes were retrieved, citrate buffer pro ving efficient for seven. PAb 240 epitope was restored by target unmas king fluid only. No false positivity was observed. Fixation-induced lo ss of p53 immunoreactivity was minimal for formalin (two of 10 tumors for one antibody each), more significant for Bouin (six of 10 tumors f or one to five antibodies). On the other hand, staining intensity was maintained or even improved, and nonspecific staining reduced, through fixation. We conclude that p53 stabilization can be detected on routi nely processed archival tumor samples with a reliability similar to th at of frozen tissue by means of a microwave-based procedure and a pane l of at least three antibodies, with epitopes on the N-terminal, C-ter minal, and midpart of the molecule.